Compositions and methods for reversibly inducing continual growth in normal cells

ABSTRACT

A mutant modified cyclin dependent kinase protein, or biologically active fragment, derivative, homolog or analog thereof is provided, which reversibly induces continual growth in cultured cells when administered to the cells exogenously in culture. Methods of reversibly inducing continual growth in cultured cells, and methods of screening cancer-causing agents with the continual growth-induced cells, are also provided.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisional patent application serial No. 60/334,760, filed on Nov. 15, 2001.

FIELD OF THE INVENTION

[0002] This invention relates to compositions and methods for inducing a reversible immortalized phenotype in normal cultured cells. In particular, the invention relates to inducing this phenotype with a mutant modified form of cyclin dependent protein kinase.

BACKGROUND OF THE INVENTION

[0003] Growth and division of living cells involve a regular series of events and processes that comprise the cell cycle. The cell cycle is typically described as a set of phases separated by gaps. There are two main phases; the M phase (the time when the cell is dividing) and the interphase (the time when the cell is not dividing). The interphase is subdivided into the time when DNA synthesis is proceeding, known as the S- or synthesis phase, and the gaps that separate the S-phase from mitosis.

[0004] The M phase consists of nuclear division (mitosis) and cytoplasmic division (cytokinesis). The process of cytokinesis terminates the M phase and marks the beginning of the interphase of the next cell cycle. The daughter cells resulting from completion of the M phase thus begin the interphase of a new cell cycle.

[0005] G1 is the gap after mitosis, but before DNA synthesis starts. G2 is the gap after DNA synthesis is complete, before mitosis begins. Interphase is thus composed of G1, S-phase and G2, and normally comprises 90% or more of the total cell cycle time.

[0006] Appropriate growth and differentiation of a cell depends on an orderly progression through the cell cycle. This progression is controlled by a series of positive and negative regulators, such as the cyclins and the cyclin dependent kinases, which act at defined points in the cycle. Processes such as regulated proliferation, differentiation, senescence and apoptosis depend on the proper functioning and interaction of these cell cycle regulators.

[0007] For example, the point in G1 at which cells irrevocably commit to DNA synthesis (and thus enter the cell cycle) is controlled by protein complexes consisting of cyclin dependent kinases cdk4 or cdk6, and the D-type cyclins D1, D2 and D3. Association of the D-type cyclins with cdk4 or cdk6 results in catalytic activation of these cyclin dependent kinases. Activated cdk4 or cdk6 in turn phosphorylates the retinoblastoma (Rb) family of proteins.

[0008] The Rb proteins negatively regulate the passage of cells from G1 to S-phase by sequestering transcription factors such as E2F, which are critical to the G1/S transition. Phosphorylation of the Rb proteins by activated cdk4 or cdk6 inactivates the Rb proteins, and prevents the sequestration of the transcription factors. The cell then progresses through the G1 block into S-phase.

[0009] The cdk4 cyclin dependent kinase is regulated by members of the INK4 family of proteins, in particular the p16^(Ink4a) kinase inhibitor. p16^(Ink4a) appears to associate with cdk4 and prevents the phosphorylation of Rb proteins. Mutations in the p16^(Ink4a) protein abolish the ability of p16^(Ink4a) to associate with cdk4 and block Rb phosphorylation. Such mutations have been implicated in familial melanomas. Ranade et al. (1995), Nat. Gen. 10: 114-116. Deletions and rearrangements in human chromosome 9p21 encompassing the p16^(Ink4a) gene have been associated with various forms of cancer. Noburi et al. (1994), Nature 368: 753-756; Kamb et al. (1994), Science 264: 436-440.

[0010] A mutation in the p16^(Ink4a) binding site on cdk4 has also been identified in patients with familial melanoma. The missense mutation results in an exchange of arginine for cysteine at position 24 of the cdk4 protein, with a consequent loss of affinity for p16^(Ink4a). The mutated cdk4, called cdk4^(R24C), is still able to bind cyclin D1 and form a functional kinase. See Wolfel et al. (1995), Science 269: 1281-1284 and Zuo et al. (1996), Nat. Gen. 12: 97-99.

[0011] Transgenic mice homozygous for the cdk4^(R24C) mutation exhibit an increase in weight of 5-10% as compared to control littermates, an increased population of testicular Leydig cells, and pancreatic islet hyperplasia. However, these mice do not develop melanoma similar to that observed in humans carrying the same cdk4^(R24C) mutation. Rane et al. (1999), Nat. Gen. 22: 44-52.

[0012] cdk6 is also inhibited by members of the p16 protein family. A mutation in the p16 protein binding site of cdk6 which converts Arg31 to Cys has been reported in a neuroblastoma cell line; see Easton J et al. (1998), Cancer Res. 58: 2624-2632. This exchange of Arg for Cys reduces binding of p16 inhibitor protein to cdk6.

[0013] Progression through the G1 block into the S-phase of the cell cycle is also affected by the cyclin dependent protein kinase cdk2. cdk2 is active when phosphorylated on threonine 160 upon association with either cyclin E (which is expressed during late G1) or cyclin A (which is expressed during S-, G2- and M-phases). Cyclin A or E/cdk2 complexes can be inactivated by phosphorylation on Thr14 or Tyr15 or when associated with inhibitory proteins such as p27KIP1.

[0014] It is well known that cells grown in culture experience cell cycle arrest, for example through contact inhibition when the cells become confluent in the culture vessel. Growth-arrested cells can by stimulated to divide by removing them from the culture vessel and seeding them at a lower density into new culture vessels. This process is known as “passaging” the cells. After a given number of passages, certain cell types can undergo replicative senescence and cease growing altogether, regardless of the cell density in the culture vessel. Replicative senescence is a significant problem where large numbers of cells or numerous passages are desired. Still other cell types are difficult to grow in culture at all, regardless of the passage number or cell density.

[0015] Therefore, it is sometimes desirable to deliberately perturb the cell cycle of cultured cells, so that the cells do not undergo cell cycle arrest, replicative senescence or apoptosis. Ideally, the cells would exhibit an immortalized phenotype; that is, would maintain constant proliferation rates without displaying any morphological features of senescing cells. The deliberately “immortalized” cells would continuously progress through successive cell cycles, and generate large numbers of cells and high passage numbers. For example, one may wish to induce the immortalized phenotype in order to grow large numbers of cells for experimentation or transplantation. Likewise, one may wish to grow large numbers of cells that produce, either naturally or by design, a compound of therapeutic or commercial interest.

[0016] However, the continuously growing cells should not exhibit a transformed (i.e., cancerous) phenotype, especially if the cells are to be used therapeutically. The reversibly “immortalized,” but non-transformed, cells can also be used as a background for testing possible carcinogens. It is therefore desirable to induce a state of continuous growth in the cultured cells that is not permanent. It is also desirable that the reversible state of continuous growth is inducible without extensive manipulation of the cultured cells. Techniques which require extensive manipulation (e.g., transfection by nucleic acids) are expensive, increase the risk of culture contamination, and are often ineffective. Moreover, the expression of exogenous genetic material in cultured cells is notoriously difficult to control.

[0017] Continuously growing cells, especially cells of higher mammals, can experience shortening of chromosome telomeres with each cell cycle. Eventually, this shortening reaches critical levels and causes the cell to undergo replicative senescence and cell death by apoptosis. Cell death can occur even though the cells have been induced to progress continuously through the cell cycle. Thus, to proliferate beyond this senescent checkpoint, telomere length must be restored and maintained above a threshold level. In some embryonic stem cells and tumor cells, telomere length is preserved by an enzyme called telomerase. There is some evidence that expressing the gene for TERT, which encodes the catalytic subunit of telomerase, can induce telomere lengthening in telomerase-negative cells. See Hodes R J (1999), J. Exp. Med. 190: 153-156 and Urquidi et al. (2000), An. Rev. Med. 51: 65-79.

[0018] There is thus a need for compositions and methods which induce a state of continuous growth in cultured cells without extensive manipulation of cells, wherein the state of continuous growth is not a permanent characteristic of the cells but may be reversed easily and at will.

SUMMARY OF THE INVENTION

[0019] It has now been found that cdk4 or cdk6 proteins, or biologically active fragments, derivatives, homologs or analogs thereof, that contain mutations which prevent binding of inhibitor proteins can induce a reversible state of continual growth in cultured cells when administered exogenously to the cells in culture.

[0020] It has also been found that cdk2 proteins, or biologically active fragments, derivatives, homologs or analogs thereof, that contain mutations which prevent cdk2 inactivation while allowing binding with cyclins A or E can induce a reversible state of continual growth in cultured cells when administered exogenously to the cells in culture.

[0021] Thus, the invention provides a continual growth-inducing composition comprising at least one compound comprising a cdk2, cdk4 or cdk6 protein, or biologically active fragment, derivative, homolog or analog thereof, having an activating mutation and one or more modifications which allow the compound to enter a cell when administered exogenously to a cell in culture. The one or more modifications can comprise a leader sequence which directs entry of the compound into the cell. The composition can additionally comprise a compound comprising the catalytic subunit of telomerase, or a biologically active fragment, derivative, homolog or analog thereof, which also has one or more modifications which allow it to enter a cell when administered exogenously to a cell in culture. Preferably, the leader sequence is designed so that it is cleaved from the compound inside the cell.

[0022] The invention also provides a method of inducing a reversible state of continuous growth in cultured cells, comprising the steps of:

[0023] providing a culture of viable cells;

[0024] contacting the cells with an effective amount of a composition comprising at least one compound comprising a cdk2, cdk4 or cdk6 protein, or biologically active fragment, derivative, homolog or analog thereof, having an activating mutation and one or more modifications which allow the compound to enter the cells, so that a state of continuous growth is induced for as long as the cells are in contact with the composition; and

[0025] optionally reversing the state of continuous growth by removing the compound from contact with the cells.

[0026] The invention provides a method of screening an agent for the ability to transform cultured cells, comprising the steps of:

[0027] providing a culture of viable cells;

[0028] contacting the cells with an effective amount of a composition comprising at least one compound comprising a cdk2, cdk4 or cdk6 protein, or biologically active fragment, derivative, homolog or analog thereof, having an activating mutation and one or more modifications which allow the compound to enter the cells, so that a state of continuous growth is induced for as long as the cells are in contact with the composition;

[0029] contacting the cells with an agent; and

[0030] evaluating the cells for the presence of a transformed phenotype.

[0031] In one embodiment, the agent to be evaluated comprises a chemical carcinogen, mutagen or teratogen. In another embodiment, the agent to be evaluated comprises nucleic acid sequences encoding, for example, a potential oncogene.

[0032] The invention also provides a cultured cell in which a reversible state of continual growth has been induced by a composition comprising at least one compound comprising a cdk2, cdk4 or cdk6 protein, or biologically active fragment, derivative, homolog or analog thereof having an activating mutation and one or more modifications which allow the compound to enter the cell when administered exogenously.

Amino Acid Abbreviations

[0033] The nomenclature used to describe the peptide compounds of the present invention follows the conventional practice wherein the amino group is presented to the left and the carboxy group to the right of each amino acid residue. In the formulae representing selected specific embodiments of the present invention, the amino-and carboxy-terminal groups, although not specifically shown, will be understood to be in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by a one-letter or three-letter designation, corresponding to the trivial name of the amino acid, in accordance with the following schedule: A Alanine Ala C Cysteine Cys D Aspartic Acid Asp E Glutamic Acid Glu F Phenylalanine Phe G Glycine Gly H Histidine His I Isoleucine Ile K Lysine Lys L Leucine Leu M Methionine Met N Asparagine Asn P Proline Pro Q Glutamine Gln R Arginine Arg S Serine Ser T Threonine Thr V Valine Val W Tryptophan Trp Y Tyrosine Tyr

Definitions

[0034] The following definitions, of terms used throughout the specification, are intended as an aid to understanding the scope and practice of the present invention.

[0035] By “cdk4 activating mutation” or “cdk6 activating mutation” is meant a mutation in a cdk4 or cdk6 protein or biologically active fragment, homolog, derivative or analog thereof, which prevents binding of an inhibitor (e.g., a p16 protein, such as p16^(Ink4a) or an equivalent inhibitor) but does not affect cdk4 or cdk6 kinase activity.

[0036] By “cdk2 activating mutation” is meant a mutation in a cdk2 protein or biologically active fragment, homolog, derivative or analog thereof, which prevents phosphorylation of cdk2 at Thr14 or Tyr15, and/or prevents binding of cdk2 to inhibitory proteins such as p27KIP1, but which does not affect cdk2 kinase activity.

[0037] “Biologically active”, when referring to a cdk2, cdk4 or cdk6 protein, or a fragment, derivative, homolog or analog thereof, means the ability to induce continual growth as measured by the cell culture assay of Example 4 below.

[0038] “Biologically active”, when referring to a TERT protein, or a fragment, derivative, homolog or analog thereof, means the ability to extend or maintain telomeric sequences as measured by the telomeric repeat amplification protocol (TRAP) assay described in Example 9 below.

[0039] The expression “amino acid” as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids. “Standard amino acid” means any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source. As used herein, “synthetic amino acid” also encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the peptides of the present invention, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the peptide's circulating half life without adversely affecting their activity. Additionally, a disulfide linkage can be present or absent in the peptides of the invention.

[0040] Amino acids have the following general structure:

[0041] Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.

[0042] “Amino-terminal truncation fragment” with respect to an amino acid sequence means a fragment obtained from a parent sequence by removing one or more amino acids from the amino-terminus thereof.

[0043] As used herein, “protecting group” with respect to a terminal amino group refers to a terminal amino group of a peptide, which terminal amino group is coupled with any of various amino-terminal protecting groups traditionally employed in peptide synthesis. Such protecting groups include, for example, acyl protecting groups such as formyl, acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethane protecting groups such as benzyloxycarbonyl; and aliphatic urethane protecting groups, for example, tert-butoxycarbonyl or adamantyloxycarbonyl. See Gross and Mienhofer, eds., The Peptides, vol. 3, pp. 3-88 (Academic Press, New York, 1981) for suitable protecting groups.

[0044] As used herein, “protecting group” with respect to a terminal carboxy group refers to a terminal carboxyl group of a peptide, which terminal carboxyl group is coupled with any of various carboxyl-terminal protecting groups. Such protecting groups include, for example, tert-butyl, benzyl or other acceptable groups linked to the terminal carboxyl group through an ester or ether bond.

[0045] “Carboxy-terminal truncation fragment” with respect to an amino acid sequence means a fragment obtained from a parent sequence by removing one or more amino acids from the carboxy-terminus thereof.

[0046] “Derivative” includes any purposefully generated peptide which in its entirety, or in part, has a substantially similar amino acid sequence to a protein. Derivatives can be characterized by single or multiple amino acid substitutions, deletions, additions, or replacements. These derivatives can include (a) derivatives in which one or more amino acid residues of a protein are substituted with conservative or non-conservative amino acids, (b) derivatives in which one or more amino acids are added to a protein, (c) derivatives in which one or more of the amino acids of a protein includes a substituent group, (d) derivatives in which a protein or a portion thereof is fused to another peptide (e.g., serum albumin), (e) derivatives in which one or more nonstandard amino acid residues (i.e., those other than the 20 standard L-amino acids commonly found in naturally occurring proteins) are incorporated or substituted into the a protein sequence, and (f) derivatives in which one or more nonamino acid linking groups are incorporated into or replace a portion of a protein.

[0047] “Fragment” refers to a portion of the a protein amino acid sequence comprising at least two amino acid residues, and which retains biological activity. Fragments can be generated by amino-terminal truncation, carboxy-terminal truncation or both of these. Fragments can also be generated by chemical or enzymatic digestion or peptide synthesis.

[0048] “Homolog” includes any nonpurposely generated peptide which in its entirety, or in part, has a substantially similar amino acid sequence to a protein and has an activating mutation. Homologs can include paralogs, orthologs, and naturally occurring alleles or variants of a protein.

[0049] By “libraries” is meant pools and subpools of pro-analogs.

[0050] “Peptide” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which can comprise a protein's or peptide's sequence. The amino acids of the peptides described herein and in the appended claims are understood to be either D or L amino acids with L amino acids being preferred.

[0051] “Variant” as the term is used herein, is a nucleic acid or peptide that differs from a reference nucleic acid or peptide respectively, but retains essential properties. Changes in the sequence of a nucleic acid variant might not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or can result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or it can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides can be made by mutagenesis techniques or by direct synthesis.

[0052] As used herein, a peptide or a portion of a peptide which has a “substantially similar amino acid sequence” to a reference protein means the peptide, or a portion thereof, has an amino acid sequence identity or similarity to the reference protein of greater than about 80%. Preferably, the sequence identity is greater than about 85%, more preferably greater than about 90%, particularly preferably greater than about 95%, and most preferably greater than about 98%. Amino acid sequence similarity or identity can be computed by using the BLASTP and TBLASTN programs which employ the BLAST (basic local alignment search tool) 2.0.14 algorithm; BLASTP and TBLASTN settings to be used in such computations are indicated in Table 1 below. Amino acid sequence identity is reported under “Identities” by the BLASTP and TBLASTN programs. Amino acid sequence similarity is reported under “Positives” by the BLASTP and TBLASTN programs. Techniques for computing amino acid sequence similarity or identity are well known to those skilled in the art, and the use of the BLAST algorithm is described in Altschul et al. (1990), J. Mol. Biol. 215: 403-10 and Altschul et al. (1997), Nucleic Acids Res. 25:3389-3402, the disclosures of which are herein incorporated by reference in their entirety. BLASTP and TBLASTN programs utilizing the BLAST 2.0.14 algorithm and can be accessed through the National Center for Biotechnology Information website maintained by the National Institutes of Health and the National Library of Medicine. TABLE 1 Settings to be used for the computation of amino acid sequence similarity or identity with BLASTP and TBLASTN programs utilizing the BLAST 2.0.14 algorithm. Expect Value 10 Filter Low complexity filtering using SEG program* Substitution Matrix BLOSUM62 Gap existence cost 11 Per residue gap cost 1 Lambda ratio 0.85 Word size 3

[0053] “Substantially similar nucleic acid sequence” means a nucleic acid sequence corresponding to a reference nucleic acid sequence wherein the corresponding sequence encodes a peptide having substantially the same structure and function as the polypeptide encoded by the reference nucleic acid sequence; e.g., where only changes in amino acids not affecting the polypeptide function occur. Preferably, the substantially similar nucleic acid sequence encodes the peptide encoded by the reference nucleic acid sequence. The percentage of identity between the substantially similar nucleic acid sequence and the reference nucleic acid sequence is, for example, at least 80%. Preferably, the sequence identity is at least about 85%, more preferably at least about 90%, particularly preferably at least about 95%, most preferably at least about 99%. Substantial similarity of nucleic acid sequences can be determined by comparing the sequence identity of two sequences, for example by physical/chemical methods (i.e., hybridization) or by sequence alignment via computer algorithm. Suitable nucleic acid hybridization conditions to determine if a nucleic acid sequence is substantially similar to a reference nucleic acid sequence are: 1) for low stringency—50% formamide; 5×Standard Saline Citrate (SSC) (20×SSC=3M NaCl; 0.3 M sodium citrate 2H₂O; pH 7.0); 20 mM sodium phosphate; 5× Denhardt's solution (100× Denhardt's solution=10 g Ficoll 400; 10 g polyvinylpyrollidone; 10 g bovine serum albumin fraction V; H₂O to 100 ml); 10% sodium dextran sulfate; 100 micrograms/ml denatured salmon sperm DNA; rotating at 37° C. overnight in a hybridization oven, with washing 3× for 20 min. in 2×SSC; 0.1% sodium dodecyl sulfate (SDS) at 37° C., followed by washing 3× in 0.1% SSC; 0.1% SDS at 37° C.; 2) for medium stringency—50% formamide; 5×SSC; 20 mM sodium phosphate; 5× Denhardt's solution; 10% sodium dextran sulfate; 100 micrograms/ml denatured salmon sperm DNA; rotating at 42° C. overnight in a hybridization oven, with washing 3× for 20 min. in 2×SSC; 0.1% sodium dodecyl sulfate (SDS) at 42° C., followed by washing 3× in 0.1% SSC; 0.1% SDS at 42° C.; 3) for high stringency—50% formamide; 5×SSC; 20 mM sodium phosphate; 5× Denhardt's solution; 10% sodium dextran sulfate; 100 micrograms/ml denatured salmon sperm DNA; rotating at 55° C. overnight in a hybridization oven, with washing 3× for 20 min. in 2×SSC; 0.1% sodium dodecyl sulfate (SDS) at 55° C., followed by washing 3× in 0.1% SSC; 0.1% SDS at 55° C. Suitable computer algorithms to determine substantial similarity between two nucleic acid sequences include, but are not limited to: GCS program package (Devereux et al. (1984), Nucl. Acids Res. 12: 387), and the BLASTN, FASTA programs (Altschul et al. (1990), J. Molec. Biol. 215: 403) or “BLAST 2 Sequences” alignment program available through the National Center for Biotechnology Information website maintained by the National Institutes of Health and the National Library of Medicine.

[0054] The default settings provided with these programs are adequate for determining substantial similarity of nucleic acid sequences for purposes of the present invention.

[0055] “Substantially purified” refers to a population of peptides or cells which is substantially homogenous in character due to the removal of other compounds (e.g., other peptides, nucleic acids, carbohydrates, lipids) or other cells originally present. “Substantially purified” is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which might be present, for example, due to incomplete purification, addition of stabilizers, or formulation into a pharmaceutically acceptable preparation.

[0056] “Synthetic mutant” includes any purposefully generated mutant or variant derived from a protein. Such mutants can be purposefully generated by, for example, chemical mutagenesis, polymerase chain reaction (PCR) based approaches, or primer based mutagenesis strategies well known to those skilled in the art.

[0057] “Transfection” is the introduction of a nucleic acid into a cell.

[0058] “Transformed” or “transformation” refers to the induction of a phenotype in cultured cells characterized at least by non-reversible, uncontrolled growth, the ability to form foci, and attachment-independent growth.

[0059] A “nucleic acid sequence” is a linear segment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention, the nucleic acid molecule is preferably a segment of DNA.

[0060] A “gene sequence” is a nucleic acid sequence capable of directing expression of a particular nucleic acid sequence in an appropriate host cell, comprising a promoter operably linked to the nucleic acid sequence of interest which is operably linked to termination signals. A gene sequence also typically comprises nucleic acid sequences required for proper translation of the expressed nucleic acid sequence. The gene sequence can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The gene sequence can also be one which is naturally occurring but has been obtained in a recombinant form useful for expression in cultured cells. Typically, however, the gene sequence is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transfection event. The expression of the gene sequence can be under the control of a constitutive promoter, or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus.

[0061] “Operatively linked” refer to two nucleic acid sequences that are related physically or functionally. For example, a promoter or regulatory DNA sequence is said to be “operatively linked” to DNA sequence that produces an RNA or encodes a protein if the two sequences are operatively linked, or situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.

[0062] A “promoter” is an untranslated nucleic acid sequence upstream of the coding region that contains the binding site for RNA polymerase and initiates transcription of RNA. The promoter region can also include other regulatory elements.

[0063] “Regulatory elements” refer to sequences involved in controlling the expression of a gene sequence. Regulatory elements comprise a promoter operably linked to the nucleic acid sequence of interest and termination signals. They also typically encompass sequences required for proper translation of a gene sequence.

BRIEF DESCRIPTION OF THE FIGURES

[0064] FIGS. 1A and 1B—Cell Growth Characteristics of cdk4^(R24C/R24C) Mouse Embryonic Fibroblasts (MEFs). FIG. 1A: 3×10⁵ MEF cells (passage<4) from cdk4^(+/+) (open circles), cdk4^(+/R24C) (open squares) and cdk4^(R24C/R24C) (closed circles) mice were grown for the indicated time in days, and the number of viable cells were counted using trypan blue exclusion analysis. FIG. 1B: 3×10⁵ MEF cells (passage<4) from cdk4^(+/+) (+/+, open bars), cdk4^(+/R24C) (+/R24C, hatched bars) and cdk4^(R24C/R24C) (R24C/R24C, solid bars) mice were exponentially cultured for 24 hours and the percentage of cells in G0/G1, S and G2/M were determined by FACs analysis upon staining with propidium iodide.

[0065]FIG. 2—Escape From Senescence and Immortalization in cdk4^(R24C/R24C) MEFs. cdk4^(+/+) (open circles; n=4), cdk4^(+/R24C) (open squares; n=4) and cdk4^(R24C/R24C) (closed circles; n=4) MEF cultures were propagated in DMEM media supplemented with 10% FBS for the indicated passages according to the 3T3 protocol. The graph shows the accumulated number of doublings that representative cultures have undergone during 20 successive passages.

DETAILED DESCRIPTION OF THE INVENTION

[0066] The present invention provides a composition which, when added exogenously to cultured cells, reversibly induces continual cell growth. The continual cell growth is induced for as long as the composition is in contact with the cultured cells.

[0067] The continual growth-inducing composition of the invention comprises at least one compound comprising a cdk2, cdk4 or cdk6 cyclin dependent kinase protein, or a biologically active fragment, derivative, homolog or analog thereof, that has an activating mutation. The compounds of the invention are therefore constitutively active kinases inside a cultured cell. The compounds can further comprise modifications which allow rapid and efficient transport into the cultured cell by simply contacting the compounds with the cells, so that extensive manipulations of the cultured cells are not required.

[0068] As used herein, “cdk4 protein” or “cdk6 protein” includes the cdk4 or cdk6 protein from any species. cdk4 and cdk6 cDNA sequences from various species are listed in Table 2. One of ordinary skill can readily identify nucleic acid sequences from other species as encoding cdk4 or cdk6 proteins, based on similarity to the sequences listed in Table 2. Nucleic acid sequences that exhibit substantial similarity to the Table 2 sequences can be considered as encoding cdk4 or cdk6 proteins, and can be used to derive compounds comprising a continual growth-inducing composition according to the invention, as described in detail below. Extant proteins can also be identified as cdk4 or cdk6 proteins by comparison to the protein sequences listed in Table 2. TABLE 2 cdk4 and cdk6 cDNA and Protein Sequences Encoded GenBank Acc. Protein Species No.¹ SEQ ID NO: cdk4(wt²) Homo sapiens ³ XM053138 protein 1 cDNA 2 cdk4(wt) Homo sapiens ³ XM048541 protein 3 cDNA 4 cdk4(wt) Homo sapiens ³ XM048543 protein 5 cDNA 6 cdk4(wt) Mus musculus NM009870 protein 7 (Balb/C) cDNA 8 cdk4(wt) Rattus norvegicus L11007 protein 9 (rat) cDNA 10 cdk4(wt) Sus scrofa U68478 protein 11 (domestic pig) cDNA 12 cdk4(wt) Xenopus laevis X89477 protein 13 (African clawed frog) cDNA 14 cdk4^(R24C) Homo sapiens Z48970 protein 15 (mutant) cDNA 16 cdk6 Homo sapiens XM_004987 protein 17 cDNA 18 cdk6 Homo sapiens protein 19 (mutant) cDNA 20 cdk6 Mus musculus AF132483 protein 21 (Balb/C) cDNA 22 cdk6 Rattus norvegicus AF352168 protein 23 (rat) cDNA 24 cdk6 Gallus gallus L77991 protein 25 (chicken) cDNA 26

[0069] The primary amino acid sequences of the allelic variants of normal human cdk4 protein are given in SEQ ID NOS: 1, 3 and 5. Normal human cdk4 is 303 amino acids long, and comprises a p16^(Ink4a) binding site which includes the Arg residue at position 24 (Arg2⁴).

[0070] A preferred activating mutation is the exchange of the conserved Arg residue for Cys in the p16^(Ink4a) binding site of cdk4. This activating mutation, called “cdk4^(R24C)” in humans, is described by Wolfel et al. (1995), Science 269: 1281-1284 and Zuo et al. (1996), Nat. Gen. 12: 97-99, the entire disclosures of which are herein incorporated by reference. The primary amino acid sequence of cdk4^(R24C) is given in SEQ ID NO: 15.

[0071] Other mutants of the cdk4 protein which have similar biological activity to cdk4^(R24C) (i.e., cdk4 activating mutations) can be generated by random or site-directed mutagenesis, protein engineering, recombinant DNA technology, or a combination of these techniques.

[0072] One or more specific cdk4 amino acids for mutation may be chosen, based on the known sequence, structure and function of the cdk4 or cdk4^(R24C) protein. For example, inspection of the cdk4 amino acid sequences encoded by the cDNAs listed in Table 2 shows that this protein is highly conserved among diverse species. Because of this conservation, one skilled in the art can readily identify amino acids in any cdk4 protein which, when mutated, would be expected to produce activating mutations.

[0073] For example, each listed cdk4 protein (with the exception of mutant cdk4^(R24C)) has a conserved Arg residue at the p16^(Ink4a) binding site (position 24 in the human, mouse, rat and pig; position 27 in X. laevis). Thus, exchanging the conserved Arg at the p16^(Ink4a) binding site for a Cys or another amino acid residue, analogous to the human cdk4^(R24C) mutation, would result in an activating mutation in the cdk4 protein from any species.

[0074] Comparison of, for example, the X. laevis and human cdk4 sequences shows that a number of amino acid residues around the conserved Arg are also highly conserved. Sequence comparisons suitable for identifying conserved cdk4 sequences can be performed, for example, with publicly available alignment algorithms such as the “BLAST 2 Sequences” alignment program described above.

[0075] A conserved amino acid sequence around the conserved Arg residue at the cdk4 protein p16^(Ink4a) binding site, as identified by BLAST 2 Sequence alignment, is: (SEQ ID NO: 27) Tyr-Glu-Pro-Val-Ala-Glu-Ile-Gly-Val-Gly-Ala-Tyr- Gly-Thr-Val-Tyr-Lys-Ala-Arg-Asp-Xaa₁-Xaa₂-Ser-Gly- Xaa₃-Phe-Val-Ala-Leu-Lys-Xaa₄-Val-Arg-Val.

[0076] The conserved Arg residue is underlined, and Xaa₁, Xaa₂, Xaa₃ and Xaa₄ represent any amino acid. Preferably, Xaa₁ is Leu or Pro; Xaa₂ is Glu or His; Xaa₃ is Lys or His; and/or Xaa₄ is Asn or Ser. It can be seen in the forgoing preferred amino acid pairs that the choice between Asn or Ser for Xaa₄ represents a conservative substitution. Without wishing to be bound by any theory, the sequence conservation around the conserved Arg residue in SEQ ID NO: 27 indicates the importance of the conserved residues in p16^(Ink4a) binding. One of ordinary skill in the art can readily choose one or more of the amino acids in SEQ ID NO: 27 for mutation, in order to produce a mutation in any cdk4 protein that prevents binding of inhibitor protein but allows kinase activity (i.e., an activating mutation). The cell culture assay described in Example 4 below can be used to confirm that a given mutation in this conserved sequence results in a cdk4 activating mutation.

[0077] The primary amino acid sequence of the normal human cdk6 protein is given in SEQ ID NO: 17. Normal human cdk6 is 326 amino acids long, and comprises a conserved Arg residue in a p16 inhibitory protein binding site, analogous to cdk4 Arg24, at position 31 (Arg31). A preferred cdk6 activating mutation is the exchange of the conserved Arg residue for Cys in cdk6, as described in Easton J et al. (1998), Cancer Res. 58: 2624-2632, the disclosure of which is herein incorporated by reference.

[0078] Comparison of the human and chicken cdk6 sequences in Table 2 shows that all of the amino acid residues around the conserved Arg in the p16 protein binding site are conserved. Sequence comparisons suitable for identifying conserved cdk6 sequences can be performed, for example, with publicly available alignment algorithms such as the “BLAST 2 Sequences” alignment program described above.

[0079] A conserved amino acid sequence around the conserved Arg residue at the cdk6 proteins, as identified by BLAST 2 Sequence alignment, is: (SEQ ID NO: 28) Tyr Glu Cys Val Ala Glu Ile Gly Glu Gly Ala Tyr Gly Lys Val Phe Lys Ala Arg Asp Leu Lys Asn Gly Gly Arg Phe Val Ala Leu Lys Arg Val Arg Val.

[0080] The conserved Arg residue is underlined. Without wishing to be bound by any theory, the sequence conservation around the conserved Arg residue in SEQ ID NO: 28 indicates the importance of the conserved residues in p16 inhibitor protein binding. One of ordinary skill in the art can readily choose one or more of these amino acids for mutation in order to produce a mutation in any cdk6 protein that prevents inhibitor binding but allows kinase activity (i.e., an activating mutation). The cell culture assay described in Example 4 below can be used to confirm that a mutation in this conserved sequence results in a cdk6 activating mutation.

[0081] Amino acids in cdk4 or cdk6 proteins which can be mutated to produce activating mutations can also be identified by comparison of the human cdk4 and cdk6 sequences around their respective conserved Arg residues. Sequence comparisons suitable for identifying conserved cdk6 sequences can be performed, for example, with publicly available alignment algorithms such as the “BLAST 2 Sequences” alignment program described above.

[0082] A conserved amino acid sequence around the conserved Arg residues in the cdk4 and cdk6 proteins, as identified by BLAST 2 Sequence alignment, is: (SEQ ID NO: 29) Tyr Glu Xaa₁ Val Ala Glu Ile Gly Xaa₂ Gly Ala Tyr Gly Xaa₃ Val Xaa₄ Lys Ala Arg Asp Xaa₅ Xaa₆ Xaa₇ Gly Xaa₈ Phe Val Ala Leu Lys Xaa₉ Val Arg Val.

[0083] The conserved Arg residue is underlined, and Xaa₁ through Xaa₉ represent any amino acid. Preferably, Xaa₁ is Cys or Pro; Xaa₂ is Glu or Val; Xaa₃ is Lys or Thr; Xaa₄ is Phe or Tyr; Xaa₅ is Lys or Pro; Xaa₆ is Asn or His; Xaa₇ is Gly or Ser; Xaa₈ is Arg or His; and/or Xaa₉ is Arg or Ser. It can be seen in the forgoing preferred amino acid pairs that the choices for Xaa₄, Xaa₅, Xaa₇, Xaa₈, and Xaa₁₀ represent conservative substitutions. Without wishing to be bound by any theory, the sequence conservation around the conserved Arg residue in SEQ ID NO: 29 indicates the importance of the conserved residues in constructing activating mutations. One of ordinary skill in the art can readily choose one or more of these amino acids for mutation in order to produce a mutation that prevents inhibitor binding but allows kinase activity in any cdk4 or cdk6 protein (i.e., an activating mutation). The cell culture assay described in Example 4 below can be used to confirm that a mutation in this conserved sequence results in a cdk4 or cdk6 activating mutation.

[0084] As used herein, “cdk2 protein” includes the cdk2 protein from any species. cdk2 cDNA and protein sequences from various species are listed in Table 3. One of ordinary skill can readily identify nucleic acid sequences from other species as encoding cdk2 proteins, based on similarity to the sequences listed in Table 3. Nucleic acid sequences that exhibit substantial similarity to the Table 3 sequences can be considered as encoding cdk2 proteins, and can be used to derive compounds used in a continual growth-inducing composition according to the invention, as described in detail below. Extant proteins can also be identified as cdk2 proteins by comparison to the protein sequences listed in Table 3. TABLE 3 cdk2 cDNA and Protein Sequences Encoded GenBank Protein Species Acc. No.¹ SEQ ID NO: cdk2 Homo sapiens XM_049152 Protein 30 cDNA 31 cdk2 Homo sapiens N/A² Protein 32 (mutant) cDNA 33 cdk2 Mus musculus NM_016756 Protein 34 (mouse) cDNA 35 cdk2L Mus musculus AJ223732 Protein 36 (mouse) cDNA 37 cdk2 Mesocricetus auratus D17350 Protein 38 (golden hamster) cDNA 39 cdk2L Mesocricetus auratus D17351 Protein 40 (golden hamster) cDNA 41 cdk2 Cricetulus griseus AJ223949 Protein 42 (Chinese hamster) cDNA 43 cdk2L Cricetulus griseus AJ223952 Protein 44 (Chinese hamster) cDNA 45 cdk2-alpha Rattus norvegicus D28753 Protein 46 (rat) cDNA 47 cdk2-beta Rattus norvegicus D63162 Protein 48 (rat) cDNA 49 cdk2 Carassius auratus S40289 Protein 50 (goldfish) cDNA 51 cdk2 Sphaerechinus granularis AJ224917 Protein 52 (sea urchin) cDNA 53 cdk2 Xenopus laevis U07979 Protein 54 (African clawed frog)

[0085] The primary amino acid sequence of the normal human cdk2 protein is given in SEQ ID NO: 30. Normal human cdk2 is 298 amino acids long, and comprises a conserved Arg residue at position 22 (Arg22) analogous to cdk4 Arg24 and cdk6 Arg31. A suitable cdk2 activating mutation is the exchange of the conserved Arg residue for Cys in humans. In mice, rats, hamster X. laevis (African clawed frog) and Carassius auratus (goldfish), the conserved Arg is replaced by a Lysine at position 22. In these species, a longer “beta” form of cdk2, comprising 346 amino acids, has been observed (see Table 3). The beta form of cdk2 still has the conserved Lys at position 22. Thus, another activating mutation is the exchange of Lys22 for Cys in the alpha or beta form of cdk2 from these species.

[0086] Other activating mutations comprise alterations in the known binding site for inhibitory proteins such as p27KIP1, or alterations in the amino acid residues which are phosphorylated to inactivate cdk2; i.e., Thr 14 and Tyr15.

[0087] Comparison of the human cdk2 and human cdk4 sequences shows that a number of amino acid residues around the conserved Arg are also highly conserved. Sequence comparisons suitable for identifying conserved cdk2 and cdk4 sequences can be performed, for example, with publicly available alignment algorithms such as the “BLAST 2 Sequences” alignment program described above.

[0088] A conserved amino acid sequence around the conserved Arg residue at the human cdk2 and cdk4 proteins, as identified by BLAST 2 Sequence alignment, is: (SEQ ID NO: 55) Xaa₁ Xaa₂ Xaa₃ Val Xaa₄ Xaa₅ Ile Gly Xaa₆ Gly Xaa₇ Tyr Gly Xaa₈ Val Tyr Lys Ala Arg Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Gly Xaa₁₃ Xaa₁₄ Val Ala Leu Lys Xaa₁₅ Xaa₁₆ Arg.

[0089] The conserved Arg residue is underlined, and Xaa₁ through Xaa₁₆ represent any amino acid. Preferably, Xaa₁ is Phe or Pro; Xaa₂ is Glu or Gln; Xaa₃ is Lys or Pro; Xaa₄ is Glu or Ala; Xaa₅ is Lys or Glu; Xaa₆ is Glu or Val; Xaa₇ is Thr or Ala; Xaa₈ is Val or Thr; Xaa₉ is Asn or Asp; Xaa₁₀ is Lys or Pro; Xaa₁₁ is Leu or His; Xaa₁₂ is Thr or Ser; Xaa₁₃ is Glu or His; Xaa₁₄ is Val or Phe; Xaa₁₅ is Lys or Ser; and/or Xaa₁₆ is Ile or Arg. It can be seen in the forgoing preferred amino acid pairs that the choices for Xaa₁, Xaa₂, Xaa₉, Xaa₁₂, Xaa₁₃, and Xaa₁₆ represent conservative substitutions. Without wishing to be bound by any theory, the sequence conservation around the conserved Arg residue in SEQ ID NO: 55 indicates the importance of the conserved residues in generating cdk2 activating mutations. One of ordinary skill in the art can readily choose one or more of these amino acids for mutation in order to produce a mutation that prevents inhibitor binding but allows kinase activity in any cdk2 protein (i.e., an activating mutation). The cell culture assay described in Example 4 below can be used to confirm that a mutation in this conserved sequence results in a cdk2 activating mutation.

[0090] Amino acids in cdk2, cdk4 or cdk6 proteins which can be mutated to produce activating mutations can also be identified by other means. For example, the three-dimensional structure of the proteins can be determined by protein X-ray crystallography and/or multidimensional NMR spectroscopy. These spectrographic techniques can be coupled with computer algorithms which can model how changes in the amino acid residues might affect the structure and function of the proteins (see, for example, Nilsson et al. (1992), Curr. Opin. in Struc. Biol. 2: 569-575; Presta (1992), Curr. Opin. in Struc. Biol 2: 593-596; Cech (1992), Curr. Opin. in Struc. Biol. 2: 605-609; and Pickersgill and Goodenough (1991), Trends in Food Sci. and Tech. 5: 122-126). Alternatively, modeling algorithms alone can be used without spectrographic studies to identify amino acid residues important for cdk2, cdk4 or cdk6 protein function. In particular, the regions surrounding the conserved Arg or Lys residue discussed above can be targeted for modification.

[0091] Thus, it is understood that the present invention encompasses not only continual growth-inducing compounds comprising the cdk4^(R24C) protein sequence, but also other mutants of cdk2, cdk4 or cdk6 that exhibit biological activity similar to cdk4^(R24C), particularly with respect to the ability to reversibly induce continual growth in cultured cells.

[0092] Having established an amino acid(s) in cdk2, cdk4 or cdk6 to be modified, an altered nucleic acid sequence can be prepared which encodes a protein with the desired amino acid change. The mutant protein itself can also be synthesized directly from amino acid substrates, as described more filly below. However, the present discussion will focus on generating proteins via recombinant nucleic acid technology.

[0093] A nucleic acid sequence containing the desired alterations in the cdk2, cdk4 or cdk6 protein can be synthesized de novo, or an unaltered nucleic acid sequence can be synthesized or isolated for subsequent mutagenesis. The nucleic acid sequence can then be subcloned into the appropriate vector for propagation in an appropriate host.

[0094] The subcloned nucleic acid sequences can then be expressed directly to generate the altered protein, or (if beginning with the unaltered sequence) subjected to site directed mutagenesis. Suitable nucleic acid coding sequences for producing mutated cdk2, cdk4 or cdk6 proteins with activating mutations are given in Tables 2 and 3 above. One of ordinary skill can readily identify other nucleic acid sequences which encode cdk2, cdk4 or cdk6 proteins based on similarity to the sequences listed in Tables 2 and 3. Nucleic acid sequences that exhibit substantial similarity to the sequences of Tables 2 and 3 can be considered cdk2, cdk4 or cdk6 protein coding sequences, and can be used to derive continual growth-inducing compounds according to the present invention.

[0095] Intracellularly produced cdk2, cdk4 or cdk6 protein mutants can be obtained from the host cell by cell lysis, or by using heterologous signal sequences fused to the protein which cause secretion of the protein into the surrounding medium. Preferably, the signal sequence is designed so that it can be removed by chemical or enzymatic cleavage, as described below for the PTD sequences. The proteins thus produced can then be purified by affinity chromatography utilizing tags incorporated into the construct including, but not limited to, 6×His, GST or Myc.

[0096] The techniques used to transform cells, construct vectors, construct oligonucleotides, perform site-specific mutagenesis, and the like are widely practiced in the art, and most practitioners are familiar with the standard resource materials which describe specific conditions and procedures. However, the following discussion is presented as a guideline for generating cdk2, cdk4 or cdk6 proteins with activating mutations, according to the present invention. All documents cited in the following discussion are incorporated by reference.

[0097] Both prokaryotic and eukaryotic systems can be used to express the nucleic acid sequences encoding the cdk2, cdk4 or cdk6 protein mutants. Prokaryotic hosts are preferred, for example various strains of E. coli. However, other microbial strains can also be used. Plasmid vectors which contain replication sites, selectable markers and regulatory sequences derived from a species compatible with the host are preferred.

[0098] Particularly preferred are bacterial plasmid expression systems which utilize regulatory systems compatible with E. coli cells. For example, E. coli can be transformed using derivatives of pBR322, a plasmid derived from an E. coli species by Bolivar et al. (1977), Gene 2: 95. Plasmid pBR322 contains genes for ampicillin and tetracycline resistance, and thus provides multiple selectable markers which can be either retained or destroyed in constructing the desired vector. Other suitable plasmid vectors include plasmids pUC9-TSF11 and pUC9delH3-pTSF-3. These plasmids are derived from pUC9 (Messing and Vieira (1982), Gene 19: 259-268), which contains parts of pBR322.

[0099] Commonly used prokaryotic regulatory sequences suitable for constructing plasmid vectors include bacterial promoters for transcription initiation, optionally with an operator, and ribosome binding site sequences. Commonly used promoters include the lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al. (1977), Nature 198: 1056); the tryptophan (trp) promoter system (Goeddel et al. (1980), Nucl. Acids Res. 8: 4057); the lambda-derived P_(L) promoter (Shimatake et al. (1981), Nature 292: 128); and the trp-lac (trc) promoter system (Amann and Brosius (1985), Gene 40: 183).

[0100] In addition to bacteria, eukaryotic microbes such as yeast can also be used as hosts. Laboratory strains of Saccharomyces cerevisiae (Baker's yeast) are preferred, although a number of other strains or species are commonly available. Vectors employing, for example, the 2μ origin of replication described in Broach (1983), Meth. Enz. 101: 307, or other yeast compatible origins of replication (see, for example, Stinchcomb et al. (1979), Nature 282: 39; Tschumper et al. (1980), Gene 10: 157; and Clarke et al. (1983), Meth. Enz. 101: 300) can be used. Regulatory sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes (see Hess et al. (1968), J. Adv. Enzyme Reg. 7:149 and Holland et al. (1978), Biochemistry 17: 4900). Additional promoters known in the art include the promoter for 3-phosphoglycerate kinase (Hitzeman et al. (1980), J. Biol. Chem. 255: 2073). Other suitable yeast promoters, which have the additional advantage of transcription controlled by growth conditions and/or genetic background, include the promoter regions for alcohol dehydrogenase 2; isocytochrome C; acid phosphatase; degradative enzymes associated with nitrogen metabolism; the alpha factor system; and enzymes responsible for maltose and galactose utilization.

[0101] For yeast hosts, it is also believed that terminator sequences are desirable at the 3′ end of the coding sequences. Such terminators are found in the 3′ untranslated region following the coding sequences in yeast-derived genes.

[0102] It is also possible to express nucleic acid sequences in eukaryotic host cell cultures derived from multicellular organisms. See, for example, U.S. Pat. No. 4,399,216 of Axel et al. These systems have the ability to splice out introns, and thus can be used directly to express genomic fragments. For example, a genomic sequence for human cdk4 is found in GenBank record Ace. No. U81031, for pig cdk4 is found in GenBank record Acc. No. U68478, and for X. laevis cdk2 is found in GenBank record Acc. No. U07979, the disclosures of which are herein incorporated by reference. However, non-genomic (e.g., cDNA) sequences can also be expressed.

[0103] Useful mammalian host cell lines for expressing mutant cdk2, cdk4 or cdk6 protein according to the present invention include VERO, HeLa, baby hamster kidney (BHK), CV-1, COS (e.g., COS-7), MDCK, NIH 3T3, L, and Chinese hamster ovary (CHO) cell lines. Expression vectors for such cells ordinarily preferably comprise promoters and regulatory sequences compatible with mammalian cells such as, for example, the SV40 early and late promoters (Fiers et al. (1978), Nature 273: 113), or other viral promoters such as those derived from polyoma, Adenovirus 2, bovine papilloma, or avian sarcoma viruses. The controllable promoter hMTII (Karin et al. (1982), Nature 299: 797-802) can also be used.

[0104] Depending on the host cell used, transfection of the plasmid vector is accomplished using standard techniques appropriate to the cell. The calcium treatment employing calcium chloride, as described by Cohen (1972), Proc. Natl. Acad. Sci. USA 69: 2110, or the RbCl₂ method described in Maniatis et al., Molecular Cloning: A Laboratory Manual (1982), Cold Spring Harbor Press, p. 254 and Hanahan (1983), J. Mol. Biol. 166: 557-580, can be used for prokaryotes or other cells which contain substantial cell wall barriers. For cells without such cell walls (i.e., eukaryotic; for example mammalian cells), the calcium phosphate precipitation method of Graham and van der Eb (1978), Virology 52: 546, optionally as modified by Wigler et al. (1979), Cell 16: 777-785 can be used. Transformations into yeast can be carried out according to the method of Beggs (1978), Nature 275: 104-109.

[0105] Construction of suitable vectors for a given host, containing the desired coding and suitable regulatory sequences, involves standard ligation and restriction techniques which are well understood in the art. Isolated plasmids, nucleic acid sequences, or synthesized oligonucleotides are cleaved, tailored, and re-ligated in the form desired.

[0106] The desired nucleic acid coding sequence for insertion into a plasmid vector can be retrieved from available cDNA or genomic DNA libraries, or from available plasmids. Preferred coding sequences are SEQ. ID NO: 1 and SEQ. ID. NO: 2. Alternatively, the desired nucleic acid coding sequence can be synthesized in vitro starting from the individual nucleoside derivatives. For example, nucleic acid sequences of sizeable length, e.g., 500-1000 bp, can be prepared by synthesizing individual overlapping complementary oligonucleotides and filling in single stranded non-overlapping portions using DNA polymerase in the presence of the deoxyribonucleotide triphosphates. This approach has been used successfully in the construction of several genes of known sequence. See, for example, Edge (1981), Nature 292: 756; Nambair et al. (1984), Science 223: 1299; and Jay (1984), J. Biol. Chem. 259: 6311.

[0107] Synthetic nucleic acid sequences can be prepared by, for example, the phosphotriester method as described in Edge et al., supra, and Duckworth et al. (1981), Nucl. Acids Res. 9: 1691; or the phosphoramidite method as described in Beaucage and Caruthers (1981), Tet. Letts. 22: 1859 and Matteucci and Caruthers (1981), J. Am. Chem. Soc. 103: 3185. The nucleic acid sequences can also be prepared using commercially available automated oligonucleotide synthesizers.

[0108] Typically, synthetic nucleic acids are provided single-stranded sequences. It is often desirable to phosphorylate the 3′ end of the single-stranded nucleic acid sequences prior to annealing with complementary sequences, for example to facilitate linking of nucleic acid fragments to form larger sequences. Phosphorylation of single stranded nucleic acid sequences prior to annealing can be accomplished, for example, using an excess (e.g., approximately 10 units) of polynucleotide kinase to 1 nanomole substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl₂, 5 mM dithiothreitol, 1-2 mM ATP, 0.1 mM spermidine, and 0.1 mM EDTA. If it is desired to radiolabel the nucleic acid sequences, 1.7 pmoles [λ³²P]-ATP (2.9 mCi/mmole) can be added to the reaction cocktail.

[0109] Other components for constructing suitable plasmid vectors are available, typically carried in other plasmids. These components can be excised from their source plasmids and ligated together with the nucleic acid sequence of interest, using standard restriction and ligation procedures.

[0110] Site specific nucleic acid cleavage, or restriction, is generally performed by treating nucleic acid sequences with suitable restriction enzyme(s) under conditions well-known in the art. Moreover, suitable reaction conditions for a given restriction enzyme are typically specified by the manufacturer of commercially available restriction enzymes. See, e.g., New England Biolabs Product Catalog, 2001.

[0111] In general, about 1 microgram of plasmid or nucleic acid sequence is cleaved by one unit of restriction enzyme in about 20 microliters of buffered solution. An excess of restriction enzyme is often used to insure complete digestion of the nucleic acid substrate with incubation times of about one hour to two hours at the optimum temperature for each enzyme as described by the manufacturer.

[0112] After each incubation, restriction enzyme can be inactivated and removed from the nucleic acid sequence by extraction with phenol/chloroform, optionally followed by ether extraction, and the nucleic acid recovered from aqueous fraction by precipitation with 2 to 2½ volumes of ethanol. If desired, size separation of the cleaved nucleic acid fragments can be performed by polyacrylamide or agarose gel electrophoresis using standard techniques. A general description of size separation techniques is found in Methods in Enzymology (1980), 65: 499-560.

[0113] Many restriction enzymes leave single-stranded overhangs after cleavage of nucleic acid sequences. Nucleic acid fragments with single-stranded overhangs can be ligated with sequences containing complementary overhangs (so called “sticky-end” ligation) or, if there is no overhang, can be ligated with any other blunt-ended nucleic acid sequences.

[0114] Nucleic acid fragments can be “blunt-ended” by, for example, incubation with the large fragment of E. coli DNA polymerase I (Klenow fragment) in the presence of the four deoxynucleotide triphosphates (dNTPs), using incubation times of about 15 to 25 min. at 20 to 25° C. in 50 mM Tris pH 7.6, 50 mM NaCl, 6 mM MgCl₂, 6 mM DTT and 0.1-1.0 mM dNTPs. The Klenow fragment fills in 5′ single-stranded overhangs, but “chews back” protruding 3′ single strands. After treatment with Klenow fragment, the reaction mixture containing the blunt-ended nucleic acid fragments is extracted with phenol/chloroform and ethanol precipitated. Treatment under appropriate conditions with S1 nuclease or BAL-31 results in hydrolysis of any remaining single-stranded portions.

[0115] Ligation of nucleic acid sequences can be performed in 15-50 microliter volumes under the following standard conditions and temperatures, for example, 20 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 10 mM DTT, 33 microgram/ml BSA, 10 mM-50 mM NaCl, and either 40 micromolar ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for “sticky end” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C. (for “blunt end” ligation). Intermolecular “sticky end” ligations are typically performed at 33-100 micrograms/ml total DNA concentrations (5-100 nM total end concentration). Intermolecular blunt end ligations are typically performed at 1 micromolar total ends concentration.

[0116] To avoid unwanted self-ligation of the vector, fragments of nucleic acids used for vector construction are commonly treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase (CIP) in order to remove the 5′ phosphates. Phosphatase reactions are typically conducted at pH 8 in approximately 10 mM Tris-HCl, 1 mM EDTA using about 1 unit of BAP or CIP per microgram of vector, at 60° C. for about one hour. Phosphatased nucleic acid fragments are recovered by extraction with phenol/chloroform and ethanol precipitation as described above.

[0117] To verify correct construction of the plasmid vector, plasmids are transfected into a suitable host, amplified, extracted, and analyzed by sequence and/or restriction analysis as is known in the art. For example, any E. coli strain (e.g., MC1061 described in Casadaban et al. (1980), J. Mol. Biol. 138: 179-207) or other suitable host can be transfected with the finished plasmid according to known techniques. Successful transfectants are selected by ampicillin, tetracycline or other antibiotic resistance (or with other appropriate markers), as is understood in the art.

[0118] Plasmids can be extracted from the transfectants according to known methods, for example the method of Clewell et al. (1969), Proc. Natl. Acad. Sci. (USA) 62: 1159, optionally following chloramphenicol amplification (see Clewell (1972), J. Bacteriol. 110: 667). See also Holmes et al. (1981), Anal. Biochem. 114: 193-197 and Birnboim et al. (1979), Nucl. Acids Res. 7: 1513-1523. Commercially available nucleic acid “mini-preps” can also be used, such as are available from Quiagen, Boehringer Mannheim, Stratagene, Invitrogen, and others (see DeFrancesco L (1997), The Scientist 11: 22 for a description of commercially available plasmid preparation kits and their suppliers).

[0119] Isolated plasmid can be analyzed, for example, by hybridization to appropriate radiolabeled probes in a “dot blot” analysis (e.g., as described by Kafatos et al. (1977), Nucl. Acid Res. 7: 1541-1552); Southern hybridization analysis (e.g., as described by Southern (1975), J. Mol. Biol. 98: 503-517); restriction enzyme analysis; or by nucleic acid sequencing (e.g., via the dideoxy nucleotide method of Sanger et al. (1977), Proc. Natl. Acad. Sci. (USA) 74: 5463, as further described by Messing et al. (1981), Nucl. Acids Res. 9: 309, or the method of Maxam et al. (1980), Methods in Enzymology 65: 499).

[0120] Mutants of the cdk2, cdk4 or cdk6 proteins can be directly expressed from plasmids constructed as described above, if the nucleic acid coding sequence in the plasmid already contains sequence modification sufficient to produce the desired amino acid change(s). Alternatively, plasmids containing unmodified cdk2, cdk4 or cdk6 protein coding sequences can be subjected to site-directed mutagenesis to produce the desired sequence changes. Mutant proteins can then be expressed from the mutagenized plasmids.

[0121] A preferred method for generating mutants of cdk2, cdk4 or cdk6 proteins that exhibit activating mutations is site-directed mutagenesis. This technique can be used to target precisely the location and type of modification desired; for example, a single base in a nucleic acid sequence can be changed to any of the other three bases by means of oligonucleotide primers. The nucleic acid can then encode a totally different amino acid, resulting in a mutagenized protein (see McPherson, M. J., Directed Mutagenesis, Oxford Univ. Press, NY (1991); Carter, P., Biochem. J. 237: 1-7 (1986); and Nickoloff and Deng (1992), Anal. Biochem. 200: 81-88). Nucleic acid sequences for site-directed mutagenesis can be provided with appropriate regulatory elements suitable for any host, including bacteria, yeast, or eukaryotic cells. Exemplary regulatory elements and hosts are discussed in more detail below.

[0122] Techniques for site-directed mutagenesis are well-known in the art. Such techniques generally involve inducing nucleotide base changes or deletions at a specific codon in the coding sequence of interest, using a synthetic primer which omits or alters a codon so that it codes for another amino acid. It is apparent that when deletions are introduced, the proper reading frame for the coding sequence must be maintained for expression of the desired protein.

[0123] For example, the methods of Zoller and Smith (1982), Nucl. Acids Res. 10: 6487-6500 or Adelman et al. (1983), DNA 2: 183-193 can be used. These methods involve annealing a synthetic oligonucleotide primer carrying limited base mis-matches to a stretch of single stranded phage DNA carrying the coding sequence to be mutagenized.

[0124] The mutagenizing primer can be hybridized to the genome of a single-stranded bacteriophage such as M13 or phi-X174, into which a single-stranded portion of the nucleic acid coding sequence has been cloned. The source of the single-stranded nucleic acid coding sequence is preferably a plasmid vector as described above. It will be appreciated that the phage can carry either the sense strand or antisense strand of the gene. Mutagenizing primers are complementary to the coding strand sequence carried by the phage, except for a limited, preferably single-base, mismatch which defines a desired codon change.

[0125] Techniques for designing mutagenizing primers of the appropriate size and sequence to achieve the desired changes are known in the art. For example, relevant factors in designing primers for use in oligonucleotide-directed mutagenesis (e.g., extent of mismatch, overall primer size, size of portions flanking the mutation site, etc.) are described by Smith and Gillam (1981), in Genetic Engineering: Principles and Methods, Plenum Press 3: 1-32. The specific changes to be made in the coding sequence are chosen beforehand, based on sequence and structure-function analyses described above.

[0126] In general the overall length of the mutagenizing primer will be such as to optimize stable, selective hybridization at the mutation site, with the 5′ and 3′ extensions from the mutation site being of sufficient size to avoid editing of the mutation by the exonuclease activity of DNA polymerase. Mutagenizing primers in accordance with the present invention can contain from about 18 to about 45 bases, preferably from about 23 to about 27 bases, with at least about three bases extending 3′ from the mutation site.

[0127] The mutagenizing primer is hybridized to the phage genome carrying the coding sequence to be altered, for example according to the conditions described by Smith and Gillam, supra. Hybridization temperature can range between about 0° C. and about 70° C., preferably between about 10° C. to about 50° C. After hybridization, the primer is extended on the phage DNA by reaction with DNA polymerase I, T₄ DNA polymerase, or other suitable DNA polymerase. The resulting linear double-stranded DNA is then converted to closed circular double-stranded DNA by treatment with a DNA ligase such as T₄ DNA ligase. Any remaining single-stranded DNA molecules can be destroyed by S1 endonuclease treatment.

[0128] The resulting closed circular double-stranded DNA is transfected into a phage-supporting host bacterium. Cultures of the transfected bacteria are plated in top agar, permitting plaque formation from single cells which harbor the phage. Theoretically, 50% of the new plaques will contain phage having, as a single strand, the altered coding sequence, and 50% will have the original coding sequence. Plaques containing the altered coding sequence are hybridized with labeled nucleic acid probe containing a region exactly complementary to the altered sequence. The hybridization conditions are stringent enough to permit only annealing of the probe only to an exact match; mismatches with the original strand are sufficient to prevent probe hybridization. Plaques showing hybridization with the probe are then picked, cultured, and the DNA recovered. The recovered DNA can then be used to express the mutant protein.

[0129] Other site-directed mutagenesis methods are available which do not require the generation of single-stranded bacteriophage template, but rather can be performed with double-stranded plasmid DNA. For example, the method of Weiner et al. (1995), Molecular Biology: Current Innovations and Future Trends (Griffin A M and Griffin H G, eds.), Horizon Scientific Press, Norfold, UK can be used. This method involves the use of the polymerase chain reaction (PCR) to incorporate site-specific mutations into virtually any double-stranded plasmid, thus eliminating the need for M13-based vectors or single-stranded rescue. Plasmids as described above comprising cdk2, cdk4 or cdk6 protein coding sequences are suitable for use in this method.

[0130] Alterations on the coding sequences can be introduced via one or both PCR primers. Mutagenizing primers can be designed using well-known techniques, based upon a predetermined amino acid change. Generally, primers for PCR-based site-directed mutagenesis will be approximately 12-24 bases long, and have a G-C content of about 45-60%. Preferably, the primers will avoid stretches of A's or T's greater than 3 bases long, and have a G or C as the 3′-most base to act as a stabilizing “clamp” during annealing with the target sequence.

[0131] Plasmid DNA strands are separated during the PCR denaturing step, allowing efficient polymerization of the mutagenizing PCR primers to the plasmid template and subsequent extension of the primers around the entire plasmid.

[0132] In order to reduce expansion of any undesired mutations, the template concentration is typically increased approximately 1000-fold over conventional PCR conditions, and the number of cycles is reduced from about 25-30 to about 5-10. As the goal is to generate a PCR product encompassing the full length of the plasmid template, heat-stable polymerases capable of synthesizing large fragments (e.g., up to 10 kb) are preferably used. A suitable polymerase is Taq Extender™ from Stratagene.

[0133] As Taq DNA polymerases tend to extend the newly synthesized DNA strand beyond the template, the PCR reactions are treated with Pfu DNA polymerase to remove the Taq DNA polymerase-extended base(s) on the linear PCR product.

[0134] Also, DNA isolated from almost all common strains of E. coli is Dam-methylated at the sequence 5′-GATC-3′. To remove “parental” DNA containing the unaltered sequence from the reaction mixture, the restriction endonuclease DpnI (which recognizes this sequence where the A residue is methylated) is therefore added after the PCR step. The DpnI treatment digests the methylated parental template and hybrid DNA molecules consisting of a methylated parent strand and a newly synthesized strand. The DpnI- and Pfu-treated linear PCR products are then circularized with T₄ DNA ligase and transfected into an appropriate bacterial host for expression of the altered protein.

[0135] A suggested protocol for PCR-based site directed mutagenesis is given in Table 4. TABLE 4 PCR-based Site Directed Mutagenesis Plasmid template DNA (approximately 0.5 picomole) is added to a PCR cocktail containing, in 25 microliter of 1× mutagenesis buffer: (20 mM Tris HCl, pH 7.5; 8 mM MgCl₂; 40 microgram/ml BSA); 12-20 picomole of each primer (one of which must contain a 5-prime phosphate), 250 uM each dNTP, 2.5 U Taq DNA polymerase, 2.5 U of Taq Extender (Stratagene). The PCR cycling parameters are 1 cycle of: 4 min at 94° C., 2 min at 50 ° C. and 2 min at 72° C.; followed by 5-10 cycles of 1 min at 94° C., 2 min at 54° C. and 1 min at 72° C. (step 1). The parental template DNA and the linear DNA extended from the mutagenesis primer are treated with DpnI (10 U) and Pfu DNA polymerase (2.5 U). The reaction is incubated at 37° C. for 30 min and then transferred to 72° C. for an additional 30 min (step 2). Mutagenesis buffer (1×, 115 microliter, containing 0.5 mM ATP) is added to the DpnI-digested, Pfu DNA polymerase-treated PCR products. The solution is mixed and 10 microliter is removed to a new microfuge tube and T₄ DNA ligase (2-4 U) added. The ligation is incubated for greater than 60 min at 37° C. (step 3). The treated solution is transformed into competent E. coli (step 4).

[0136] Site-directed mutagenesis can also be performed on double-stranded plasmid DNA without using PCR. Such methods are based on the ability of certain DNA polymerases to synthesize entire plasmids using two complementary primers. A kit for performing this method is available from Stratagene (The QuikChange™ kit).

[0137] This method uses double stranded plasmids purified, for example, by miniprep or cesium chloride gradient. Plasmid DNA with the unaltered coding sequence of interest are annealed with two synthetic oligonucleotide primers containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, are extended during temperature cycling with a high-fidelity, long extending DNA polymerase such as PfuTurbo™ DNA polymerase (Stratagene).

[0138] On incorporation of the oligonucleotide primers, a mutated plasmid containing staggered nicks is generated by primer extension. The reaction mixture is then treated with DpnI to digest methylated parental DNA template and hemi-methylated hybrid DNA, thus selecting for the mutagenized plasmid. The nicked vector DNA incorporating the desired mutations is then transformed into a suitable E. coli host for expression, for example “XL1-Blue” or “XL10-Gold” cells. Materials and methods for performing the QuikChange method are available from the manufacturer.

[0139] In addition to predetermined alterations in the cdk2, cdk4 or cdk6 proteins, libraries of plasmids expressing a spectrum of random mutations can be produced. One technique for producing such libraries is termed “error-prone PCR.” In this method, random mutations are deliberately introduced into nucleic acid coding sequences during PCR through the use of error-prone DNA polymerases. The mutated DNA sequences are cloned into expression vectors, and the resulting libraries of mutant proteins are screened for the desired protein activity. Unaltered nucleic acid coding sequences suitable for use in this method can be linear (preferably double-stranded) nucleic acid molecules comprising all or part of the cdk2, cdk4 or cdk6 protein coding sequences, or can be plasmids containing these sequences prepared as described above.

[0140] Error-prone PCR methods commonly employ Taq DNA polymerase, as it lacks proofreading activity and is inherently error prone. A preferred Taq DNA polymerase is the Mutazyme™ DNA polymerase available from Stratagene. To achieve useful mutation frequencies, the error rate of Taq DNA polymerase can be further increased by employing PCR reaction buffers containing Mn⁺⁺ and unbalanced dNTP concentrations.

[0141] Different mutation frequencies can be achieved by varying the DNA template concentration. A low initial template concentration means that the target sequence undergoes greater amplification in a given number of PCR cycles. As the Taq polymerase error rate is based on the degree of target sequence amplification, higher mutation frequencies are achieved by lower initial template concentrations. Conversely, lower mutation frequencies are achieved by using higher initial template concentrations. For structure-function analyses, a low mutation rate (1-3 nucleotide changes, or 1 amino acid change per kilobase coding sequence) is typically used. In directed evolution studies, medium mutation frequencies (3-7 nucleotide changes, or 1-4 amino acid changes per kilobase coding sequence) are generally employed. Proteins with the desired activity can also be isolated from highly mutagenized libraries exhibiting 7-20 point mutations per kilobase coding sequence.

[0142] Appropriate amounts of target nucleic acid needed to achieve the desired mutation frequency (MF) are given in Table 5, adapted from Cline and Hogrefe “Randomize Gene Sequences with New PCR Mutagenesis Kit,” Stratagene (2001), 13(4): 157-162, the entire disclosure of which is herein incorporated by reference. TABLE 5 Initial Amounts of Template DNA for Desired Mutational Frequency Initial template amount^(b) approximate # mutations per kb Mutation Level 100 ng 1-2.5 low 10 ng 2-4   1 ng 3-4.5 medium 100 pg 4-5.5 10 pg 5-6.5 1 pg 6-7   Double PCR 7-13  high Triple PCR 9-20 

[0143] A kit and method for performing error prone PCR (Genemorph™) is available from Stratagene.

[0144] Mutant cdk2, cdk4 or cdk6 proteins with the desired biological activity can be identified by their ability to reversibly induce continual cell growth when added to cells in culture. For example, a mutated protein is added to a viable culture of mouse embryonic fibroblasts isolated and cultured according to the technique of Todaro and Green (1963), J. Cell. Biol. 17: 299-313. The cultured fibroblasts are then evaluated for certain morphological and biochemical changes indicative of an immortalized phenotype. Such changes including a decreased doubling time, a higher proportion of cells in the S and G2/M phases than controls, and no contact growth inhibition. This fibroblast immortalization assay is described in Example 4 below.

[0145] The continual growth-inducing compositions of the invention can additionally comprise a compound comprising the catalytic subunit of telomerase, or a biologically active derivative, homolog or analog thereof.

[0146] The catalytic subunit of telomerase, also called TERT, is a protein of 1132 amino acids that exhibits reverse transcriptase activity. Compounds comprising TERT can be expressed from nucleic acid sequences known in the art, using the techniques described above. For the purposes of the present invention, the TERT protein should retain its natural biological activity; i.e., the ability to catalyze RNA-dependent elongation of the 3′ terminus of a chromosome, and thus maintain telomere length above the critical threshold. One of ordinary skill in the art can readily determine whether a compound comprising an TERT protein, or a fragment, derivative, homolog or analog thereof, is biologically active by testing the compound for telomerase activity by known methods; for example, by the telomeric repeat amplification protocol (TRAP) assay as described Kim et al. (1994), Science 266: 2011-2015, the disclosure of which is herein incorporated by reference. Identification of TERT proteins with the TRAP assay is also disclosed in Example 9 below.

[0147] As used herein, “TERT protein” includes the TERT protein from any species. TERT nucleic acid sequences which can be used for expressing TERT proteins are given in Table 6 below One of ordinary skill can readily identify nucleic acid sequences from other species as encoding TERT proteins, based on similarity to the sequences listed in Table 6. Nucleic acid sequences that exhibit substantial similarity to the Table 6 sequences can be considered as encoding hTERT proteins. Extant proteins can also be identified as TERT proteins by comparison to the protein sequences listed in Table 6. TABLE 6 TERT cDNA and Protein Sequences GenBank Species Acc. No.1 SEQ ID NO: Xenopus laevis AAG43537 protein 56 (African clawed frog) Mesocricetus auratus AAF17334 protein 57 (golden hamster) Rattus norvegicus AF247818 protein 58 (rat) cDNA 59 Mus musculus AAC09323 protein 60 (mouse) Homo sapiens NP_003210 protein 61 Cryptosporidium parvum AY034376 protein 62 cDNA 63 Giardia intestinalis AF195121 protein 64 cDNA 65 Mus musculus AF157502 protein 66 (mouse) cDNA 67 Candida albicans AF216872 protein 68 (yeast) cDNA 69

[0148] When used as part of the continual growth-inducing composition of the present invention, the TERT proteins, or fragments, derivatives, homologs or a analogs thereof, are preferably present in equimolar amounts with the cdk2, cdk4 or cdk6 compounds described above.

[0149] The compounds comprising the continual growth-inducing composition of the invention, including the TERT compounds described above, can also comprise one or more modifications that allow transport of the compound into a cultured cell, when contacted with the cell in culture. For example, the compounds can be modified with a leader peptide sequence that directs entry of the compound into the cell, when the compound is administered exogenously in culture. Such leader sequences, also known as “protein transduction domains” or “PTDs,” are well known in the art. A PTD can be located anywhere on the continual growth-inducing compound that does not disrupt the compound's biological activity. For compounds comprising peptides, the PTD is preferably located at the N-terminal end.

[0150] It is known that bioactive, exogenous proteins and other molecules can be delivered to any mammalian cell type by linking it to a PTD. This technique is known as “protein transduction.” See Schwarze et al. (1999), Science 285: 1569-1572, the entire disclosure of which is herein incorporated by reference. Proteins ranging in size from 15 to 120 kD have been transduced into a wide variety of human and murine cell types in vitro using this method. See Nagahara et al. (1998), Nature Med. 4: 1449; Ezhevsky et al. (1997), Proc. Natl. Acad. Sci. U.S.A. 94: 10699; Lissy et al. (1998), Immunity 8: 57; and Gius et al. (1999), Cancer Res. 59: 2577, the entire disclosures of which are herein incorporated by reference.

[0151] Without wishing to be bound by a particular theory, entry of exogenously added, PTD-linked compounds into the cell during protein transduction appears to occur in a rapid, concentration-dependent fashion. Moreover, the process appears to be receptor and transporter independent, see Derossi et al. (1996), J. Biol. Chem. 271: 18188, and may directly involve the lipid bilayer component of the cell membrane. Thus, all cell types, in particular mammalian cell types, are susceptible to protein transduction. Exogenous administration of PTD-linked compounds to cultured cells results in the rapid delivery of a roughly equal amount of the compound to each cell.

[0152] The PTD can comprise any of the known PTD sequences including for example, a peptide of eleven arginine residues (SEQ ID NO: 70) or the NH₂-terminal 11-amino acid protein transduction domain from the human immunodeficiency virus TAT protein (SEQ ID NO: 71) Other suitable leader sequences include, but are not limited to, other arginine-rich sequences; e.g., 9 to 11 arginines, or six or more arginines in combination with one or more lysines or glutamines. Such leader sequences are know in the art; see, e.g., Guis et al. (1999), Cancer Res. 59: 2577-2580, the disclosure of which is herein incorporated by reference.

[0153] Preferably, the PTD is designed so that it is cleaved from the continual growth-inducing compound within the cell. Amino acid sequences susceptible to enzymatic cleavage within a cell are known in the art.

[0154] The PTD can also comprise a label (e.g., substances which are magnetic resonance active; radiodense; fluorescent; radioactive; detectable by ultrasound; detectable by visible, infrared or ultraviolet light) so that entry of the PTD-linked compound into the cells can be monitored. Suitable labels include, for example, fluorescein isothiocyanate (FITC); peptide chromophores such as phycoerythrin or phycocyanin and the like; bioluminescent peptides such as the luciferases originating from Photinus pyrali; fluorescent proteins originating from Renilla reniformi; and radionuclides such as P³², P³³, S³⁵, I¹²⁵ or I¹³¹. For example, the label can comprise an NH₂-terminal fluorescein isothiocyanate (FITC)-Gly-Gly-Gly-Gly motif that is conjugated to the PTD.

[0155] Methods of modifying PTD sequences with labels are well known to those skilled in the art. For example, methods of conjugating fluorescent compounds such as fluorescein isothiocyanate to short peptides are described in Danen et al., Exp. Cell Res., 238:188-86 (1998), the entire disclosure of which is herein incorporated by reference. Methods of producing a FITC-labeled PTD leader sequence are described in Schwarze et al. (1999), Science 285: 1569-1572 the disclosure of which is herein incorporated by reference. Methods of radiolabeling peptides with radionuclides such as ¹²⁵I and ³⁵S are disclosed by Sambrook et al. in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Second Ed., (1989), pp. 18.24-18.29, the entire disclosure of which is herein incorporated by reference.

[0156] It is understood that the compounds comprising the continual growth-inducing composition can themselves be modified with a label, as described above for the PTD sequence.

[0157] The compounds comprising the continual growth-inducing composition and the PTD sequence can be linked by any means which allows formation of a covalent bond between the PTD sequence and the compounds. Such methods are known to those of ordinary skill in the art. For example, fusion peptides comprising a compound and a PTD can be generated by any means which permits linking two or more peptide sequences, including standard recombinant nucleic acid techniques, or solid phase peptide synthesis techniques.

[0158] Kits and methods for producing fusion peptides comprising a PTD linked to a protein of interest are commercially available. For example, the TransVector™ system (Q-B10 gene) produces fusion proteins comprising the 16 amino acid Penetratin™ peptide leader sequence, which corresponds to the Drosophila antennapedia DNA binding domain. The Voyager™ system allows creation of fusion peptides comprising the V22 PTD from Herpes Simplex Virus-1. One of ordinary skill in the art is able to generate continual growth-inducing compounds of the invention linked to a suitable PTD using such kits and methods.

[0159] Alternatively, the continual growth-inducing compositions of the invention can be introduced into a cultured cell by fusion of a liposome encapsulating the compositions with the cell membrane. Techniques to encapsulate proteins in a liposome for delivery into a cell are well known in the art. For example, the ProVectin™ Protein Delivery Reagent, available from Ingenex, can be used to deliver the present compounds into cultured cells. It is understood that the compounds comprising continual growth-inducing compositions administered to cells by liposome encapsulation do not require PTDs. For purposes of the invention, encapsulation of the continual growth inducing compounds is a “modification which allows entry of the compositions into a cell.”

[0160] The compounds comprising the continual growth-inducing compositions of the invention can comprise natural or synthetic peptides produced by any known means, including synthesis by biological systems and chemical methods.

[0161] Biological synthesis of peptides is well known in the art, and includes the transcription and translation of a synthetic gene encoding cdk2, cdk4, cdk6 proteins with an activating mutation, or biologically active fragments, homologs, and derivatives thereof. Chemical peptide synthesis includes manual and automated techniques well known to those skilled in the art.

[0162] For example, automated peptide synthesis can be performed with commercially available peptide synthesizers. Biologically active fragments according to the invention can also be obtained by the digestion or fragmentation of larger natural or synthetic peptides. Techniques to synthesize or otherwise obtain peptides and peptide fragments are well known in the art.

[0163] The peptides and fragments comprising the compounds of the present invention can be synthesized de novo using conventional solid phase synthesis methods. In such methods, the peptide chain is prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence. The use of various N-protecting groups, e.g., the carbobenzyloxy group or the t-butyloxycarbonyl group; various coupling reagents e.g., dicyclohexylcarbodiimide or carbonyldimidazole; various active esters, e.g., esters of N-hydroxyphthalimide or N-hydroxy-succinimide; and the various cleavage reagents, e.g., trifluoroactetic acid (TFA), HCl in dioxane, boron tris-(trifluoracetate) and cyanogen bromide; and reaction in solution with isolation and purification of intermediates are methods well-known to those of ordinary skill in the art.

[0164] A preferred peptide synthesis method follows conventional Merrifield solid phase procedures well known to those skilled in the art. Additional information about solid phase synthesis procedures can be had by reference to Steward and Young, Solid Phase Peptide Synthesis, W. H. Freeman & Co., San Francisco, 1969; the review chapter by Merrifield in Advances in Enzymology 32:221-296, F. F. Nold, Ed., Interscience Publishers, New York, 1969; and Erickson and Merrifield, The Proteins 2:61-64 (1990), the entire disclosures of which are herein incorporated by reference. Crude peptide preparations resulting from solid phase syntheses can be purified by methods well known in the art, such as preparative HPLC. The amino-terminus can be protected according to the methods described for example by Yang et al., FEBS Lett. 272:61-64 (1990), the entire disclosure of which is herein incorporated by reference.

[0165] The compounds comprising the continual growth-inducing composition of the present invention include derivatives of cdk2, cdk4 or cdk6 proteins having an activating mutation. The techniques for obtaining these derivatives are known to persons having ordinary skill in the art and include, for example, standard recombinant nucleic acid techniques, solid phase peptide synthesis techniques and chemical synthetic techniques as described above. Linking groups can also be used to join or replace portions of cdk2, cdk4, cdk6 and other peptides. Linking groups include, for example, cyclic compounds capable of connecting an amino-terminal portion and a carboxyl terminal portion of cdk2, cdk4, or cdk6. Techniques for generating derivatives are also described in U.S. Pat. No. 6,030,942 the entire disclosure of which is herein incorporated by reference (derivatives are designated “peptoids” in the U.S. Pat. No. 6,030,942 patent). Derivatives can also incorporate labels such as are described above into their structure.

[0166] Examples of derivatives according to the present invention include, for example, synthetic variants of cdk2, cdk4 or cdk6 proteins having an activating mutation. Derivatives can also include, for example, fusion peptides in which a portion of the fusion peptide has a substantially similar amino acid sequence to cdk2, cdk4 or cdk6 proteins having an activating mutation. Such fusion peptides can be generated as described above.

[0167] The compounds comprising the continual growth-inducing compositions of the invention also include homologs of cdk2, cdk4 or cdk6 proteins having an activating mutation. Homologs have substantially similar amino acid sequence to cdk2, cdk4, cdk6 proteins and can be identified on this basis.

[0168] The compounds of the invention also include analogs of cdk2, cdk4 or cdk6 proteins having an activating mutation. The analogs of the invention can, for example, be small organic molecules capable of binding the D-type cyclins and phosphorylating the Rb proteins, or small organic molecules capable of binding cyclins A or E. Analogs can incorporate labels such as are described above into their structure.

[0169] Without wishing to be bound by a particular theory, it is believed that the present analogs comprise a structure, called a pharmacophore, that mimics the physico-chemical and spatial characteristics of cdk2, cdk4 or cdk6 proteins having an activating mutation. Consequently, pro-analogs can, for example, be designed based on variations in the molecular structure of the cdk2, cdk4 or cdk6 protein active sites or portions of cdk2, cdk4 or cdk6 proteins. The structure and probable function of the various portions of the cdk2, cdk4 or cdk6 proteins can be determined, for example, using nuclear magnetic resonance (NMR), crystallographic, or computational methods which permit the electron density, electrostatic charges or molecular structure of these peptides to be mapped, as discussed above.

[0170] Alternatively, pro-analogs of a cdk2, cdk4 or cdk6 proteins having an activating mutation can be designed, for example, by using the retrosynthetic, target-oriented, or diversity-oriented synthesis strategies described by Schreiber (2000), Science 287:1964-1969, the entire disclosure of which is herein incorporated by reference. Retrosynthetic strategies, for example, require that key structural elements in a molecule such as cdk2, cdk4 or cdk6 protein having an activating mutation be identified and then incorporated into the structure of otherwise distinct pro-analogs generated by organic syntheses. U.S. Pat. No. 6,030,942, in particular Example 4 therein, describes retrosynthetic methods for the design and selection of analogs based on key structural elements in an inhibitory peptide, and is herein incorporated by reference in its entirety (analogs are designated “peptidomimetics” in the 6,030,942 patent).

[0171] The solid-phase synthesis methods described by Schreiber supra can be used to generate a library of distinct pro-analogs generated by organic syntheses. Briefly, a suitable synthesis support, for example a resin, is coupled to a pro-analog precursor. The pro-analog precursor is subsequently modified by organic reactions such as, for example, Diels-Alder cyclization. The immobilized pro-analog can then be released from the solid substrate. Pools and subpools of pro-analogs can be generated by automated synthesis techniques in parallel, such that all synthesis and resynthesis can be performed in a matter of days. Such pools and subpools of pro-analogs are said to comprise libraries. Once generated, pro-analog libraries can be screened for analogs; i.e. compounds exhibiting one or more biological activities of cdk2, cdk4 or cdk6 proteins having an activating mutation. Analogs can thus be identified by their ability to reversibly induce continual cell growth as determined, for example, by the cell culture assay described in Example 4 below.

[0172] The continual growth-inducing compounds of the invention can additionally comprise a TERT protein, or a biologically active fragment, derivative, homolog or analog thereof. Biologically active fragments, derivatives, homologs or analogs of TERT proteins can be made as discussed above for cdk2, cdk4 and cdk6 proteins. One of ordinary skill in the art can readily determine if a fragment, derivative, homolog or analog of TERT protein is biologically active by the ability of the compound to extend or maintain telomeric sequences, for example as measured by the telomeric repeat amplification protocol (TRAP) assay described in Example 9 below.

[0173] The present invention provides methods of inducing continual growth in viable cultures of eukaryotic cells by contacting the cultured cells with an effective amount of a continual growth-inducing composition. The continual growth-inducing composition comprises at least one of the cdk2, cdk4, or cdk6 proteins having an activating mutation, or biologically active fragments, derivatives, homologs or analogs thereof, as described above. The cells experience a state of continual growth for as long as they are in contact with the continual growth-inducing composition. Optionally, the composition further comprises a TERT protein, or a biologically active fragment, derivative, homolog or analog, as described above.

[0174] Methods of contacting cultured cells with exogenous compositions are known in the art, and include administering a composition either directly to the cells, or in the culture media. Preferably, an effective amount of a continual growth-inducing composition is included in the fresh growth media which is periodically given to cells growing in culture. For example, a concentrated solution of the continual growth-inducing composition in a carrier such as sterile water, saline, growth media or the like can be diluted into fresh growth media prior to applying the fresh growth media to the cultured cells.

[0175] Any eukaryotic cell type that can be placed in culture in a viable state, for a time sufficient to allow contact with an effective amount of a continual growth-inducing composition, can be used in the present methods. Suitable culturable cell types include cells obtained from amphibians, reptiles, birds, mammals, fish, arthropods, and insects. Preferred cells are those obtained from mammals, for example humans; rodents (e.g., mice, rats and guinea pigs); rabbits; ovine mammals (e.g., sheep and goats); bovine mammals (e.g., cows); and porcine mammals (e.g., pigs). Methods for obtaining and culturing such cells are well-known in the art.

[0176] In one embodiment, the invention provides methods for inducing a state of continual growth in stem cells derived from an animal; for example, in hematopoietic stem cells, embryonic stem (ES) cells or normal blastocyst cells. Methods of obtaining embryonic stem cells and normal blastocyst cells are known in the art; see for example Thomson et al., (1998) Science 282:1145-47; and Reubinoff et al. (2000) Nat. Biotechnol. 18: 399-404, the entire disclosures of which are herein incorporated by reference. The induction of a continual state of growth in stem cells by the present methods does not lead to terminal differentiation of the stem cells. Thus, large numbers of stem cells can be produced for use in research, transplantation, etc.

[0177] Murine ES cell lines suitable for use in the present methods include the AB-1 line grown on mitotically inactive SNL76/7 cell feeder layers (McMahon and Bradley (1990), Cell 62: 1073) essentially as described (Robertson, E. J. (1987) in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E. J. Robertson, ed. (Oxford: IRL Press), p. 71-112). Other suitable murine ES lines include, but are not limited to, the E14 line (Hooper et al. (1987) Nature 326: 292-295), the D3 line (Doetschman et al. (1985) J. Embryol. Exp. Morphj. 87: 27-45), and the CCE line (Robertson et al. (1986) Nature 323: 445-448).

[0178] An extensive list of human and murine stem cell lines suitable for use in the present methods are listed in Appendix D of “Stem Cells: Scientific Progress and Future Research Directions,” report of the National Institutes of Health, 2001, the entire disclosure of which is herein incorporated by reference. The stem cells listed in the NIH report, Appendix D include hematopoietic stem cells; mesenchymal stem cells; neural stem cells; neural progenitor cells; ES cells; embryonic primordial germ cells (endoderm, mesoderm and ectoderm); skeletal muscle satellite cells; and blastocyst inner cell masses. Methods for isolating and maintaining these cells in culture are known in the art, as referenced in the NIH report, supra, Appendix D.

[0179] The effective amount of continual growth-inducing composition will vary from cell type to cell type. One of ordinary skill in the art is able to determine the effective amount for a given cell type, for example by contacting the cells with increasing concentrations of the composition and observing the morphological and growth characteristics of the cells, as outlined in Example 4 below. The concentration at which the cells exhibit the well-known characteristics of an immortalized phenotype is considered to be the minimum effective amount.

[0180] When more than one compound comprises the continual growth-inducing composition, it is preferred that the compounds are present in equimolar amounts. For most cell types, the concentration of the compounds comprising an effective amount of the continual growth-inducing composition is at most 50 nM, preferably at most 100 nM, more preferably at most 150 nM, and particularly preferably at most 300 nM. It is contemplated, however, that the compounds can be present in varying amounts relative to each other, consistent with inducing continual growth in the cultured cells.

[0181] When the cultured cells are no longer in contact with the continual growth-inducing composition, the induction of continual growth will cease. The cessation of continual growth will not be immediate, but will rather depend on the kinetics of inactivation or clearance of the continual growth-inducing composition from the cultured cells. Continual growth can be resumed, if desired, by again contacting the cells with the continual growth-inducing composition.

[0182] The present invention also provides methods of evaluating potential cancer-causing agents, such as ionizing radiation, mutagens, teratogens, carcinogens, oncogenes and the like. The method comprises contacting cultured cells reversibly induced to exhibit continual growth with potential cancer-causing agents.

[0183] As used herein, the step of contacting cultured cells with a potential cancer-causing agent includes administration of such agents exogenously in culture (e.g., by placing a chemical carcinogen, teratogen or mutagen in the culture media); the external application of ionizing radiation (e.g., by irradiation with an irradiator using ¹³⁷cesium as a source; brachytherapy devices, etc.); internal application of ionizing radiation (e.g., delivery of tritium or high-energy radionuclides to the cytoplasm or cell nucleus); or introduction of genetic material into the cell (e.g., transfection with plasmids comprising putative oncogenic sequences, infection with recombinant viruses, etc.).

[0184] For example, mutagens such as 9,10-di-methyl-1,2-benz[a]anthracene (DMBA) or tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) can be added to the culture medium in varying concentrations to determine the susceptibility of a given cell type transformation. Compounds with undetermined cancer-causing activity can also be screened with the continual growth-induced cells of the invention, in order to evaluate the carcinogenicity of the compounds.

[0185] The oncogenic potential of nucleic acid sequences can also be evaluated in the continual growth-induced cells. For example, cultured cells can be transfected with plasmids comprising gene sequences expressing suspected oncogenes, or gene sequences that carry naturally or artificially produced mutations. Methods of transfecting eukaryotic cells with nucleic acid sequences are also well known in the art, and include, for example, direct injection into the nucleus or pronucleus; electroporation; liposome transfer (e.g., with N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate, also called DOTAP); and calcium phosphate precipitation.

[0186] The transforming potential of the cancer-causing agents can be evaluated by observing the growth characteristics and morphology of the cultured cells. Transformed cells will continue to be insensitive to contact-induced growth inhibition even when the continual growth inducing compound is removed (i.e., continual growth is not reversible), and the cells will form foci in the culture vessel when cultured for extended periods. Transformed cells also exhibit characteristic morphological changes, disorganized patterns of colony growth and acquisition of anchorage-independent growth. Transformed cells also have the ability to form invasive tumors in susceptible animals, which can be assessed by injecting the cells, for example, into athymic mice or newborn animals of the same species using techniques well-known in the art.

[0187] One of ordinary skill in the art is thus able to identify a transformed phenotype in the cultured cells, and/or determine whether the cells have acquired the ability to form tumors in vivo. See, for example, Combes et al. (1999), “Cell Transformation Assays as Predictors of Human Carcinogenicity: The Report and Recommendations of ECVAM Workshop 39,” ATLA 27, 745-767, the entire disclosure of which is herein incorporated by reference.

[0188] The invention will now be illustrated with the following non-limiting examples.

EXAMPLE 1 Activating Mutations in the cdk4 Protein Lead to Increased Cell Proliferation and Shorter Cell Cycle

[0189] Mouse embryonic fibroblasts (MEFs) cultures were obtained from wild type mice (cdk4^(+/+)), transgenic mice heterozygous for the cdk4^(R24C) mutation (cdk4^(+/R24C)), and transgenic mice homozygous for the cdk4^(R24C) mutation (cdk4^(R24C/R24C)), as described in Todaro and Green (1963), J. Cell. Biol. 17: 299-313, the disclosure of which is herein incorporated by reference. The transgenic mice were generated as described in Rane et al. (1999), Nat. Gen. 22: 44-52, the disclosure of which is herein incorporated by reference. The MEFs were propagated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

[0190] MEFs (passage<4) were used for growth curve and cell cycle phase analyses. For analysis of growth curves, 3×10⁵ cells were plated in 10×2 cm, tissue culture treated polystyrene dishes (Falcon® #353003; Becton Dickinson) and the number of viable cells were counted using trypan blue exclusion analysis for four days. For analysis of cell cycle phases, 3×10⁵ cells were exponentially cultured for 24 hours in 10×2 cm, tissue culture treated polystyrene dishes (Falcon® #353003; Becton Dickinson), after which the cells were fixed in ethanol at −20° C. The percentage of cells in G0/G1, S and G2/M were determined by fluorescence-assorted cytometry (FACS) analysis upon staining for 30 min with propidium iodide after treatment with RNAse A. The results are shown in FIGS. 1a and 1 b.

[0191] The results show that cdk4^(R24C/R24C) cells grew well in culture and displayed decreased doubling times, indicating that homozygous expression of the cdk4^(R24C) protein caused acceleration of cell proliferation (FIG. 1a). The cdk4^(+/R24C) MEFs exhibited doubling times intermediate between the wild-type and cdk4^(R24C/R24C) MEFs.

[0192] To analyze the effects of cdk4^(R24C) mutation on cell cycle progression, a cell cycle analysis was performed using flow cytometry techniques. Exponentially growing cdk4^(R24C/R24C) MEFs exhibited a slightly higher proportion of cells in the S and G2M phases than either the wild-type or cdk4^(+/R24C) MEFs (FIG. 1b).

[0193] The consistent increase in the number of cdk4^(R24C/R24C) cells in the S and G2M phases, along with a concomitant decrease in the 2n population of cells (G0/G1), together with their faster growth rate (see FIG. 1a), indicates that the cdk4^(R24C/R24C) MEFs proliferate faster than the wild-type and cdk4^(+/R24C) MEFs. This seems to be due to a decrease in the lengths of the G1-phase, which results in a shorter doubling time.

[0194] As the cdk4^(+/R24C) and cdk4^(R24C/R24C) MEFs used in this Example contained one or two copies of an endogenous mutant cdk4 gene, respectively, the induction of continual growth in these cells was not reversible.

EXAMPLE 2 Mouse Embryonic Fibroblasts from a Transgenic cdk4^(R24C/R24C) Mouse Fail to Undergo Senescence and Exhibit an Immortalized Phenotype in Culture

[0195] Mouse embryonic fibroblasts (“MEFs”) were obtained from wild type mice (cdk4^(+/+)), transgenic mice heterozygous for the cdk4^(R24C) mutation (cdk4^(+/R24C)), and transgenic mice homozygous for the cdk4^(R24C) mutation (cdk4^(R24C/R24C)) as in Example 1. The MEFs were propagated in DMEM media supplemented with 10% FBS according to the 3T3 protocol (Todaro and Green, supra).

[0196] For each cell type, 3×10⁵ cells were plated per 10×2 cm, tissue culture treated polystyrene dishes (Falcon® #353003; Becton Dickinson). After 3 days, cells were trypsinized and 3×10⁵ cells from a given dish were re-plated, which constituted one passage. This process was continued for 20 successive passages and the cumulative increase in cell number was calculated according to the formula Log_((Nfinal/Ninitial))/Log 2, where Nfinal and Ninitial are the final and initial numbers of cells plated and counted after 3 days, respectively.

[0197] It is known that wild-type MEFs stop dividing after 15-30 generations, and undergo replicative senescence when cultured using the 3T3 protocol (Todaro and Green, supra). As can be seen in FIG. 2, the cdk4^(+/+) cells showed a decline in the proliferation rate at about 8-10 passages, then a short growth spurt between 10-15 passages, followed by the onset of senescence. The cdk4^(+/+) cells underwent approximately 16 population doublings during the first 20 passages in culture (FIG. 2).

[0198] Induction of senescence was confirmed by examination of cells by microscopy, where morphological changes associated with a senescence phenotype (flat, large non-dividing cells) were observed. To further confirm the phenotype of replicative senescence in the cdk4^(+/+) cells, the activity of endogenous Senescence Associated Beta-Galactosidase (SA-B-Gal), a specific biomarker of senescence, was examined as described previously (Dimri et al. (1995), Proc. Natl. Acad. Sci. USA 92:9363-7, the entire disclosure of which is herein incorporated by reference). The cdk4^(+/+) cells showed evidence of accumulated SA-B-Gal, suggestive of senescing cells.

[0199] In contrast, cdk4^(R24C/R24C) MEFs maintained constant proliferation rates and failed to display any morphological features of senescing cells as evidenced by a failure to accumulate SA-B-Gal. The cdk4^(R24C/R24C) MEFs underwent approximately 30 population doublings after 20 passages in culture (compared to 16 population doublings in control cells), indicative of a significant escape from replicative senescence.

[0200] The cdk4^(+/R24C) MEFs displayed an intermediate phenotype, with majority of the cells maintaining a constant rate of proliferation with approximately 20 population doublings in 20 passages. Approximately 50-60% of cdk4^(+R24C) MEFs displayed morphological features of senescent cells. The difference in the behavior of cdk4^(+/R24C) and cdk4^(R24C/R24C) MEFs suggests that induction of continual growth may be dependent on the levels of the mutant cdk4^(R24C) protein inside the cell. Because the cdk4^(+/R24C) and cdk4^(R24C/R24C) MEFs used in this Example contained one or two copies of an endogenous mutant cdk4 gene, respectively, the induction of continual growth in these cells was not reversible.

EXAMPLE 3 Preparation of Continual Growth Inducing Composition Comprising cdk4^(R24C)

[0201] A nucleic and sequence encoding cdk4 and the NH₂-terminal 11-amino acid PTD from the human immunodeficiency virus TAT protein (SEQ ID NO: 71) are cloned, in the same reading frame, pET-15b expression vector (Novagen) via the multicloning site by standard techniques. This vector allows production of a fusion protein with an N-terminal 6×His tag upon induction with isopropyl-thio-beta-D-thiogalactoside (IPTG). The pET-15b plasmid construct is used to transfect high-expressing bacterial strain BL21 (DE3) (Novagen) according to the manufacturer's protocols; see Novagen's “pET System Manual (9^(th) Ed.)”, May 2000, the disclosure of which is herein incorporated by reference.

[0202] One liter of transfected BL21 (DE3) cultures is induced to express the cdk4^(R24C)/PTD/His tag-fusion protein by the addition of IPTG at a final concentration of 1 mM followed by incubation at 37° C. with shaking. Cells are pelleted by centrifugation, resuspended in lysis buffer (8M urea, 100 mL NaCl, 20 mM HEPES (pH=7.0), 20 mM imidazole) and sonicated. Cell lysate is cleared and subjected to Ni-NTA (Qiagen) column chromatography according to the manufacturer's instructions, which binds the 6×His portion of the fusion protein and allows for cdk4^(R24C)/PTD/6×His-fusion protein isolation. After elution from the Ni-NTA column, the fusion protein is dialyzed against 20 mM Hepes/150 mM NaCl according to the procedure of Ezhevsky et al. (1997), Proc. Natl. Acad. Sci. USA 94: 10699-10704, the disclosure of which is herein incorporated by reference. Concentration of the dialyzed fusion protein is determined by the Bio-Rad Protein Determination Assay® (Bio-Rad) and adjusted to 1-10 micrograms/ml before filter sterilization through a 0.02 micron filter.

EXAMPLE 4 Reversible Induction of Continual Growth in Cultured Cells with the Composition of Example 3

[0203] Normal mouse embryonic fibroblasts are obtained as in Example 1 above, and are seeded in 10 ml of DMEM, 10% FBS in 10×2 cm, tissue culture treated, polystyrene dishes (Falcon® #353003, Becton Dickinson) at a cell density of 3×10⁵ cells per plate. After 24 hours, the growth media is replaced with fresh media containing of continual growth-inducing composition from Example 3 above, in various concentrations from 50 nM to 300 nM, or an equal volume of the solvent buffer for the compound. Growth media is replaced with fresh media containing the same constituents every 1 to 3 days. Population doubling is assayed as in Example 2. A significant increase in population doubling, along with absence of morphological signs of senescence indicates continual growth has been induced. Reversibility of the induced continual growth is expected on removal of the continual growth-inducing composition from contact with the cultured cells, as evaluated by the significant decrease in the population doublings of the treated cells.

EXAMPLE 5 Transformation of Continual Growth-Induced Cultured Cells with Oncogene-Containing Plasmids

[0204] The continual growth-induced cultured cells from Example 4 are transfected with pcDNA3 plasmid vectors containing the human oncogenes Ha-ras^(V12), pE1A, or c-myc. Each oncogenic plasmid is evaluated for the ability to transform the cultured cells by scoring the foci formed after 21 days in culture.

[0205] Transfection of Cultured Cells with Plasmids and Scoring of Foci

[0206] 10⁶ early passage (<4) mouse embryonic fibroblast (MEF) cells are obtained as in Example 1. The growth medium is changed 4-6 hours before transfections are begun. Transfections are performed by standard calcium phosphate or DEAE-Dextran procedures as follows.

[0207] Calcium Phosphate Transfection

[0208] The calcium phosphate transfection is performed essentially as described in Current Protocols in Molecular Biology (1996), Ausubel et al., eds., John Wiley and Sons, Inc., USA, the disclosure of which is herein incorporated by reference.

[0209] Cultured MEFs are collected and seeded at 1.5×10⁴ cells per plate (10×2 cm, tissue culture treated polystyrene dishes; Falcon® #353003, Becton Dickinson) and cultured for 18-24 hours. Approximately 5 μg of circular or linearized plasmid DNA is added to 50 microliters of 2.5M CaCl₂ and the volume is adjusted to 500 microliters with H₂O. Air is bubbled into 500 microliters of 2×HEPES buffer (0.5M HEPES, 5M NaCl, 0.5M Na₂HPO₄) in a 15 ml tube (Falcon Blue Max Jr., #352097, Becton Dickinson) while dropwise adding the 500 microliter DNA/CaCl₂ mixture. After vortexing this solution for ten seconds, the solution is added to the plated cells. After 24 hours, the cells are washed three times with sterile PBS and 10 ml of fresh media is added. Transfected cells are maintained in culture for 21 days, with fresh growth media supplied every three days.

[0210] DEAE-Dextran Transfection

[0211] DEAE-Dextran transfection is performed essentially as described in Current Protocols in Molecular Biology (1996), Ausubel et al., eds., John Wiley and Sons, Inc., USA supra. Cultured MEFs are collected and seeded at 5×10⁵ cells per plate (10×2 cm, tissue culture treated, polystyrene dishes; Falcon® #353003, Becton Dickinson) and cultured for 18-24 hours. DEAE-Dextran/DNA mix is prepared as follows: 5 ml DMEM (Gibco)/10% NuSerum (Collaborative Research), 200 microliters DNA (1-20 micrograms), 200 microliters DEAE-Dextran/chloroquine (10 mg/ml DEAE/Dextran, 2.5 mM chloroquine in sterile PBS). The plated cells are washed with PBS incubated with 10 ml of the DEAE-Dextran DNA mix for two hours. Cells are washed twice with PBS, and the growth media (DMEM, 10% FBS) is replaced. Transfected cells are maintained in culture for 21 days, with fresh growth media supplied every three days.

[0212] Scoring of Foci

[0213] At 21 days past transfection, cells are fixed and stained with Giemsa, and foci (>2 mm in diameter) as scored visually.

EXAMPLE 6 Preparation of Continual Growth Inducing Composition Comprising cdk4^(R24C) and TERT

[0214] cdk4^(R24C)/PTD/6×His fusion protein is obtained as in Example 3 above. TERT fusion protein is produced by cloning the nucleotide sequence of SEQ ID NO: 67 and the NH₂-terminal 11-amino acid PTD from the human immunodeficiency virus TAT protein (SEQ ID NO: 71), in the same reading frame, into a pET-15b expression vector (Novagen) via the multicloning site by standard techniques. This vector allows production of a fusion protein with an N-terminal 6His tag. This pET-15b plasmid construct is used to transform high-expressing bacteria BL21(DE3), and TERT/PTD/6×His fusion protein is isolated from the transfected as in Example 3, above.

[0215] The continual growth-inducing composition is made by mixing equimolar amounts of cdk4^(R24C)/PTD/6×His fusion protein and TERT/PTD/6×His fusion protein in physiological saline as a solvent buffer.

EXAMPLE 7 Reversible Induction of Continual Growth in Cultured Cells with the Composition of Example 6

[0216] Normal mouse embryonic fibroblasts are obtained as in Example 1 above, and are seeded in 10 ml of DMEM, 10% FBS in 10×2 cm, tissue culture treated, polystyrene dishes (Falcon® #353003, Becton Dickinson) at a cell density of 3×10⁵ cells per plate. After 24 hours, the growth media is replaced with fresh media containing of continual growth-inducing composition from Example 6 above in various concentrations from 50 nM to 300 nM, or an equal volume of the solvent buffer for the compound. Growth media is replaced with fresh media containing the same constituents every 1 to 3 days. Population doubling is assayed as in Example 2. A significant increase in population doubling, along with absence of morphological signs of senescence indicates continual growth has been induced. Reversibility of the induced continual growth is expected on removal of the continual growth-inducing composition from contact with the cultured cells, as evaluated by the significant decrease in the population doublings of the treated cells.

EXAMPLE 8 Preparation of Continual Growth Inducing Composition Comprising cdk4^(R24C), Mutant cdk2, Mutant cdk6, and TERT

[0217] cdk4^(R24C)/PTD/6×His fusion protein and TERT/PTD/6×His fusion protein are obtained as in Example 6 above. cdk2 and cdk6 fusion proteins having activating mutations, and including a PTD and a 6×His tag are obtained as follows.

[0218] Mutant cdk2 fusion protein is made by cloning a nucleic acid sequence encoding cdk2 with in which Arg22 is replaced by Cys (SEQ ID NO: 33) and the NH₂-terminal 11-amino acid PTD from the human immunodeficiency virus TAT protein (SEQ ID NO: 71), in the same reading frame, into a pET-15b expression vector (Novagen) via the multicloning site by standard techniques. This vector allows production of a fusion protein with an N-terminal 6His tag. This pET-15b plasmid construct is used to transform high-expressing bacteria BL21(DE3), and mutant cdk2/PTD/6×His fusion protein is isolated from the transfected as in Example 3, above.

[0219] Mutant cdk6 fusion protein is made by cloning a nucleic acid sequence encoding ckd6 in which Arg31 is replaced by Cys (SEQ ID NO: 20) and the NH₂-terminal 11-amino acid PTD from the human immunodeficiency virus TAT protein (SEQ ID NO: 71), in the same reading frame, into a pET-15b expression vector (Novagen) via the multicloning site by standard techniques. This pET-15b plasmid construct is used to transform high-expressing bacteria BL21(DE3), and mutant cdk2/PTD/6×His fusion protein is isolated from the transfected as in Example 3, above.

[0220] The continual growth-inducing composition is made by mixing equimolar amounts of cdk4^(R24C), mutant cdk2, mutant cdk6 and TERT fusion proteins obtained as describe above in physiological saline as a solvent buffer.

EXAMPLE 9 Telomeric Repeat Amplification Protocol (TRAP) Assay for Telomerase Activity of Putative TERT Compounds

[0221] A putative TERT protein, or fragment, derivative, homolog or analog thereof (hereinafter “TERT compound”) is tested for telomerase activity according to the procedure of Kim et al. (1994), Science 266: 2011-2015, the disclosure of which is herein incorporated by reference.

[0222] The TERT compound is mixed with 0.1 microgram of the non-telomeric oligonucleotide TS (5′-AATCCGTCGAGCAGAGTT-3′ (SEQ ID NO: 72)) in a 50 microliter reaction containing 20 mM Tris-HCl (pH 8.3); 1.5 mM EGTA; 50 micromolar A, C, G, T deoxynucleoside triphosphates; 1 microgram of T4g32 protein (Boehringer Mannheim); 0.1 mg/ml bovine serum albumin; 2 units of Taq DNA polymerase (Boehringer Mannheim). 0.2 to 0.4 microliter of alpha-³²P deoxyguanosine triphosphate or alpha-³²P deoxycytidine triphosphate (10 microCurie/microliter) can be added to radiolabel the extension product. The reaction mix is incubated for 10 min. at 23° C. to extend the TS oligonucleotide. CX primer (5′-(CCCTTA)₃CCCTAA-3′ (SEQ ID NO: 73)) is added, and the reaction mix is transferred to a thermal cycler for 27 rounds of PCR amplification at 94° C. for 30 s; 50° C. for 30 s; and 72° C. for 90 s. PCR products are analyzed by electrophoresis in 0.5×Tris-Borate EDTA on 15% polyacrylamide non-denaturing gels. Telomerase activity is shown by the presence of amplified telomeric repeats on the gel. Positive telomerase activity by this assay indicates that the compound being tested in an TERT compound.

[0223] All documents referred to herein are incorporated by reference. While the present invention has been described in connection with the preferred embodiments of the various figures, it is to be understood that other similar embodiments can be used or modifications and additions made to the described embodiments for performing the same function of the present invention without deviating therefrom. Therefore, the present invention should not be limited to any single embodiment, but rather should be construed in breadth and scope in accordance with the recitation of the appended claims.

1 69 1 303 PRT Homo sapiens PEPTIDE (1)...(303) human cdk4 1 Met Ala Thr Ser Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala 1 5 10 15 Tyr Gly Thr Val Tyr Lys Ala Arg Asp Pro His Ser Gly His Phe Val 20 25 30 Ala Leu Lys Ser Val Arg Val Pro Asn Gly Gly Gly Gly Gly Gly Gly 35 40 45 Leu Pro Ile Ser Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu 50 55 60 Ala Phe Glu His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr 65 70 75 80 Ser Arg Thr Asp Arg Glu Ile Lys Val Thr Leu Val Phe Glu His Val 85 90 95 Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys Ala Pro Pro Pro Gly Leu 100 105 110 Pro Ala Glu Thr Ile Lys Asp Leu Met Arg Gln Phe Leu Arg Gly Leu 115 120 125 Asp Phe Leu His Ala Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu 130 135 140 Asn Ile Leu Val Thr Ser Gly Gly Thr Val Lys Leu Ala Asp Phe Gly 145 150 155 160 Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala Leu Thr Pro Val Val Val 165 170 175 Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala 180 185 190 Thr Pro Val Asp Met Trp Ser Val Gly Cys Ile Phe Ala Glu Met Phe 195 200 205 Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Gly 210 215 220 Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro Glu Asp Asp Trp Pro Arg 225 230 235 240 Asp Val Ser Leu Pro Arg Gly Ala Phe Pro Pro Arg Gly Pro Arg Pro 245 250 255 Val Gln Ser Val Val Pro Glu Met Glu Glu Ser Gly Ala Gln Leu Leu 260 265 270 Leu Glu Met Leu Thr Phe Asn Pro His Lys Arg Ile Ser Ala Phe Arg 275 280 285 Ala Leu Gln His Ser Tyr Leu His Lys Asp Glu Gly Asn Pro Glu 290 295 300 2 1342 DNA Homo sapiens mRNA (1)...(1342) human cdk4 cDNA. 2 gtctatggtc gggccctctg cgtccagctg ctccggaccg agctcgggtg tatggggccg 60 taggaaccgg ctccggggcc ccgataacgg gccgccccca cagcaccccg ggctggcgtg 120 agggtctccc ttgatctgag a atg gct acc tct cga tat gag cca gtg gct 171 Met Ala Thr Ser Arg Tyr Glu Pro Val Ala 1 5 10 gaa att ggt gtc ggt gcc tat ggg aca gtg tac aag gcc cgt gat ccc 219 Glu Ile Gly Val Gly Ala Tyr Gly Thr Val Tyr Lys Ala Arg Asp Pro 15 20 25 cac agt ggc cac ttt gtg gcc ctc aag agt gtg aga gtc ccc aat gga 267 His Ser Gly His Phe Val Ala Leu Lys Ser Val Arg Val Pro Asn Gly 30 35 40 gga gga ggt gga gga ggc ctt ccc atc agc aca gtt cgt gag gtg gct 315 Gly Gly Gly Gly Gly Gly Leu Pro Ile Ser Thr Val Arg Glu Val Ala 45 50 55 tta ctg agg cga ctg gag gct ttt gag cat ccc aat gtt gtc cgg ctg 363 Leu Leu Arg Arg Leu Glu Ala Phe Glu His Pro Asn Val Val Arg Leu 60 65 70 atg gac gtc tgt gcc aca tcc cga act gac cgg gag atc aag gta acc 411 Met Asp Val Cys Ala Thr Ser Arg Thr Asp Arg Glu Ile Lys Val Thr 75 80 85 90 ctg gtg ttt gag cat gta gac cag gac cta agg aca tat ctg gac aag 459 Leu Val Phe Glu His Val Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys 95 100 105 gca ccc cca cca ggc ttg cca gcc gaa acg atc aag gat ctg atg cgc 507 Ala Pro Pro Pro Gly Leu Pro Ala Glu Thr Ile Lys Asp Leu Met Arg 110 115 120 cag ttt cta aga ggc cta gat ttc ctt cat gcc aat tgc atc gtt cac 555 Gln Phe Leu Arg Gly Leu Asp Phe Leu His Ala Asn Cys Ile Val His 125 130 135 cga gat ctg aag cca gag aac att ctg gtg aca agt ggt gga aca gtc 603 Arg Asp Leu Lys Pro Glu Asn Ile Leu Val Thr Ser Gly Gly Thr Val 140 145 150 aag ctg gct gac ttt ggc ctg gcc aga atc tac agc tac cag atg gca 651 Lys Leu Ala Asp Phe Gly Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala 155 160 165 170 ctt aca ccc gtg gtt gtt aca ctc tgg tac cga gct ccc gaa gtt ctt 699 Leu Thr Pro Val Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu 175 180 185 ctg cag tcc aca tat gca aca cct gtg gac atg tgg agt gtt ggc tgt 747 Leu Gln Ser Thr Tyr Ala Thr Pro Val Asp Met Trp Ser Val Gly Cys 190 195 200 atc ttt gca gag atg ttt cgt cga aag cct ctc ttc tgt gga aac tct 795 Ile Phe Ala Glu Met Phe Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser 205 210 215 gaa gcc gac cag ttg ggc aaa atc ttt gac ctg att ggg ctg cct cca 843 Glu Ala Asp Gln Leu Gly Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro 220 225 230 gag gat gac tgg cct cga gat gta tcc ctg ccc cgt gga gcc ttt ccc 891 Glu Asp Asp Trp Pro Arg Asp Val Ser Leu Pro Arg Gly Ala Phe Pro 235 240 245 250 ccc aga ggg ccc cgc cca gtg cag tcg gtg gta cct gag atg gag gag 939 Pro Arg Gly Pro Arg Pro Val Gln Ser Val Val Pro Glu Met Glu Glu 255 260 265 tcg gga gca cag ctg ctg ctg gaa atg ctg act ttt aac cca cac aag 987 Ser Gly Ala Gln Leu Leu Leu Glu Met Leu Thr Phe Asn Pro His Lys 270 275 280 cga atc tct gcc ttt cga gct ctg cag cac tct tat cta cat aag gat 1035 Arg Ile Ser Ala Phe Arg Ala Leu Gln His Ser Tyr Leu His Lys Asp 285 290 295 gaa ggt aat ccg gag tga gcaatggagt ggctgccatg gaaggaagaa 1083 Glu Gly Asn Pro Glu * 300 aagctgccat ttcccttctg gacactgaga gggcaatctt tgcctttatc tctgaggcta 1143 tggagggtcc tcctccatct ttctacagag attactttgc tgccttaatg acattcccct 1203 cccacctctc cttttgaggc ttctccttct ccttcccatt tctctacact aaggggtatg 1263 ttccctcttg tccctttccc tacctttata tttggggtcc ttttttatac aggaaaaaca 1323 aaacaaagaa ataatggtc 1342 3 303 PRT Homo sapiens PEPTIDE (1)...(303) human cdk4 3 Met Ala Thr Ser Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala 1 5 10 15 Tyr Gly Thr Val Tyr Lys Ala Arg Asp Pro His Ser Gly His Phe Val 20 25 30 Ala Leu Lys Ser Val Arg Val Pro Asn Gly Gly Gly Gly Gly Gly Gly 35 40 45 Leu Pro Ile Ser Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu 50 55 60 Ala Phe Glu His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr 65 70 75 80 Ser Arg Thr Asp Arg Glu Ile Lys Val Thr Leu Val Phe Glu His Val 85 90 95 Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys Ala Pro Pro Pro Gly Leu 100 105 110 Pro Ala Glu Thr Ile Lys Asp Leu Met Arg Gln Phe Leu Arg Gly Leu 115 120 125 Asp Phe Leu His Ala Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu 130 135 140 Asn Ile Leu Val Thr Ser Gly Gly Thr Val Lys Leu Ala Asp Phe Gly 145 150 155 160 Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala Leu Thr Pro Val Val Val 165 170 175 Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala 180 185 190 Thr Pro Val Asp Met Trp Ser Val Gly Cys Ile Phe Ala Glu Met Phe 195 200 205 Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Gly 210 215 220 Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro Glu Asp Asp Trp Pro Arg 225 230 235 240 Asp Val Ser Leu Pro Arg Gly Ala Phe Pro Pro Arg Gly Pro Arg Pro 245 250 255 Val Gln Ser Val Val Pro Glu Met Glu Glu Ser Gly Ala Gln Leu Leu 260 265 270 Leu Glu Met Leu Thr Phe Asn Pro His Lys Arg Ile Ser Ala Phe Arg 275 280 285 Ala Leu Gln His Ser Tyr Leu His Lys Asp Glu Gly Asn Pro Glu 290 295 300 4 1399 DNA Homo sapiens mRNA (1)...(1399) human cdk4 cDNA 4 ctttggcagc tggtcacatg gtgagggtgg gggtgagggg gcctctctag cttgcggcct 60 gtgtctatgg tcgggccctc tgcgtccagc tgctccggac cgagctcggg tgtatggggc 120 cgtaggaacc ggctccgggg ccccgataac gggccgcccc cacagcaccc cgggctggcg 180 tgagggtctc ccttgatctg aga atg gct acc tct cga tat gag cca gtg gct 233 Met Ala Thr Ser Arg Tyr Glu Pro Val Ala 1 5 10 gaa att ggt gtc ggt gcc tat ggg aca gtg tac aag gcc cgt gat ccc 281 Glu Ile Gly Val Gly Ala Tyr Gly Thr Val Tyr Lys Ala Arg Asp Pro 15 20 25 cac agt ggc cac ttt gtg gcc ctc aag agt gtg aga gtc ccc aat gga 329 His Ser Gly His Phe Val Ala Leu Lys Ser Val Arg Val Pro Asn Gly 30 35 40 gga gga ggt gga gga ggc ctt ccc atc agc aca gtt cgt gag gtg gct 377 Gly Gly Gly Gly Gly Gly Leu Pro Ile Ser Thr Val Arg Glu Val Ala 45 50 55 tta ctg agg cga ctg gag gct ttt gag cat ccc aat gtt gtc cgg ctg 425 Leu Leu Arg Arg Leu Glu Ala Phe Glu His Pro Asn Val Val Arg Leu 60 65 70 atg gac gtc tgt gcc aca tcc cga act gac cgg gag atc aag gta acc 473 Met Asp Val Cys Ala Thr Ser Arg Thr Asp Arg Glu Ile Lys Val Thr 75 80 85 90 ctg gtg ttt gag cat gta gac cag gac cta agg aca tat ctg gac aag 521 Leu Val Phe Glu His Val Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys 95 100 105 gca ccc cca cca ggc ttg cca gcc gaa acg atc aag gat ctg atg cgc 569 Ala Pro Pro Pro Gly Leu Pro Ala Glu Thr Ile Lys Asp Leu Met Arg 110 115 120 cag ttt cta aga ggc cta gat ttc ctt cat gcc aat tgc atc gtt cac 617 Gln Phe Leu Arg Gly Leu Asp Phe Leu His Ala Asn Cys Ile Val His 125 130 135 cga gat ctg aag cca gag aac att ctg gtg aca agt ggt gga aca gtc 665 Arg Asp Leu Lys Pro Glu Asn Ile Leu Val Thr Ser Gly Gly Thr Val 140 145 150 aag ctg gct gac ttt ggc ctg gcc aga atc tac agc tac cag atg gca 713 Lys Leu Ala Asp Phe Gly Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala 155 160 165 170 ctt aca ccc gtg gtt gtt aca ctc tgg tac cga gct ccc gaa gtt ctt 761 Leu Thr Pro Val Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu 175 180 185 ctg cag tcc aca tat gca aca cct gtg gac atg tgg agt gtt ggc tgt 809 Leu Gln Ser Thr Tyr Ala Thr Pro Val Asp Met Trp Ser Val Gly Cys 190 195 200 atc ttt gca gag atg ttt cgt cga aag cct ctc ttc tgt gga aac tct 857 Ile Phe Ala Glu Met Phe Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser 205 210 215 gaa gcc gac cag ttg ggc aaa atc ttt gac ctg att ggg ctg cct cca 905 Glu Ala Asp Gln Leu Gly Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro 220 225 230 gag gat gac tgg cct cga gat gta tcc ctg ccc cgt gga gcc ttt ccc 953 Glu Asp Asp Trp Pro Arg Asp Val Ser Leu Pro Arg Gly Ala Phe Pro 235 240 245 250 ccc aga ggg ccc cgc cca gtg cag tcg gtg gta cct gag atg gag gag 1001 Pro Arg Gly Pro Arg Pro Val Gln Ser Val Val Pro Glu Met Glu Glu 255 260 265 tcg gga gca cag ctg ctg ctg gaa atg ctg act ttt aac cca cac aag 1049 Ser Gly Ala Gln Leu Leu Leu Glu Met Leu Thr Phe Asn Pro His Lys 270 275 280 cga atc tct gcc ttt cga gct ctg cag cac tct tat cta cat aag gat 1097 Arg Ile Ser Ala Phe Arg Ala Leu Gln His Ser Tyr Leu His Lys Asp 285 290 295 gaa ggt aat ccg gag tga gcaatggagt ggctgccatg gaaggaagaa 1145 Glu Gly Asn Pro Glu * 300 aagctgccat ttcccttctg gacactgaga gggcaatctt tgcctttatc tctgaggcta 1205 tggagggtcc tcctccatct ttctacagag attactttgc tgccttaatg acattcccct 1265 cccacctctc cttttgaggc ttctccttct ccttcccatt tctctacact aaggggtatg 1325 ttccctcttg tccctttccc tacctttata tttggggtcc ttttttatac aggaaaaaca 1385 aaacaaagaa ataa 1399 5 303 PRT Homo sapiens PEPTIDE (1)...(303) human cdk4 5 Met Ala Thr Ser Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala 1 5 10 15 Tyr Gly Thr Val Tyr Lys Ala Arg Asp Pro His Ser Gly His Phe Val 20 25 30 Ala Leu Lys Ser Val Arg Val Pro Asn Gly Gly Gly Gly Gly Gly Gly 35 40 45 Leu Pro Ile Ser Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu 50 55 60 Ala Phe Glu His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr 65 70 75 80 Ser Arg Thr Asp Arg Glu Ile Lys Val Thr Leu Val Phe Glu His Val 85 90 95 Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys Ala Pro Pro Pro Gly Leu 100 105 110 Pro Ala Glu Thr Ile Lys Asp Leu Met Arg Gln Phe Leu Arg Gly Leu 115 120 125 Asp Phe Leu His Ala Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu 130 135 140 Asn Ile Leu Val Thr Ser Gly Gly Thr Val Lys Leu Ala Asp Phe Gly 145 150 155 160 Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala Leu Thr Pro Val Val Val 165 170 175 Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala 180 185 190 Thr Pro Val Asp Met Trp Ser Val Gly Cys Ile Phe Ala Glu Met Phe 195 200 205 Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Gly 210 215 220 Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro Glu Asp Asp Trp Pro Arg 225 230 235 240 Asp Val Ser Leu Pro Arg Gly Ala Phe Pro Pro Arg Gly Pro Arg Pro 245 250 255 Val Gln Ser Val Val Pro Glu Met Glu Glu Ser Gly Ala Gln Leu Leu 260 265 270 Leu Glu Met Leu Thr Phe Asn Pro His Lys Arg Ile Ser Ala Phe Arg 275 280 285 Ala Leu Gln His Ser Tyr Leu His Lys Asp Glu Gly Asn Pro Glu 290 295 300 6 1441 DNA Homo sapiens mRNA (1)...(1441) human cdk4 cDNA 6 gccctcccag tttccgcgcg cctctttggc agctggtcac atggtgaggg tgggggtgag 60 ggggcctctc tagcttgcgg cctgtgtcta tggtcgggcc ctctgcgtcc agctgctccg 120 gaccgagctc gggtgtatgg ggccgtagga accggctccg gggccccgat aacgggccgc 180 ccccacagca ccccgggctg gcgtgagggt ctcccttgat ctgaga atg gct acc 235 Met Ala Thr 1 tct cga tat gag cca gtg gct gaa att ggt gtc ggt gcc tat ggg aca 283 Ser Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala Tyr Gly Thr 5 10 15 gtg tac aag gcc cgt gat ccc cac agt ggc cac ttt gtg gcc ctc aag 331 Val Tyr Lys Ala Arg Asp Pro His Ser Gly His Phe Val Ala Leu Lys 20 25 30 35 agt gtg aga gtc ccc aat gga gga gga ggt gga gga ggc ctt ccc atc 379 Ser Val Arg Val Pro Asn Gly Gly Gly Gly Gly Gly Gly Leu Pro Ile 40 45 50 agc aca gtt cgt gag gtg gct tta ctg agg cga ctg gag gct ttt gag 427 Ser Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu Ala Phe Glu 55 60 65 cat ccc aat gtt gtc cgg ctg atg gac gtc tgt gcc aca tcc cga act 475 His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr Ser Arg Thr 70 75 80 gac cgg gag atc aag gta acc ctg gtg ttt gag cat gta gac cag gac 523 Asp Arg Glu Ile Lys Val Thr Leu Val Phe Glu His Val Asp Gln Asp 85 90 95 cta agg aca tat ctg gac aag gca ccc cca cca ggc ttg cca gcc gaa 571 Leu Arg Thr Tyr Leu Asp Lys Ala Pro Pro Pro Gly Leu Pro Ala Glu 100 105 110 115 acg atc aag gat ctg atg cgc cag ttt cta aga ggc cta gat ttc ctt 619 Thr Ile Lys Asp Leu Met Arg Gln Phe Leu Arg Gly Leu Asp Phe Leu 120 125 130 cat gcc aat tgc atc gtt cac cga gat ctg aag cca gag aac att ctg 667 His Ala Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu Asn Ile Leu 135 140 145 gtg aca agt ggt gga aca gtc aag ctg gct gac ttt ggc ctg gcc aga 715 Val Thr Ser Gly Gly Thr Val Lys Leu Ala Asp Phe Gly Leu Ala Arg 150 155 160 atc tac agc tac cag atg gca ctt aca ccc gtg gtt gtt aca ctc tgg 763 Ile Tyr Ser Tyr Gln Met Ala Leu Thr Pro Val Val Val Thr Leu Trp 165 170 175 tac cga gct ccc gaa gtt ctt ctg cag tcc aca tat gca aca cct gtg 811 Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala Thr Pro Val 180 185 190 195 gac atg tgg agt gtt ggc tgt atc ttt gca gag atg ttt cgt cga aag 859 Asp Met Trp Ser Val Gly Cys Ile Phe Ala Glu Met Phe Arg Arg Lys 200 205 210 cct ctc ttc tgt gga aac tct gaa gcc gac cag ttg ggc aaa atc ttt 907 Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Gly Lys Ile Phe 215 220 225 gac ctg att ggg ctg cct cca gag gat gac tgg cct cga gat gta tcc 955 Asp Leu Ile Gly Leu Pro Pro Glu Asp Asp Trp Pro Arg Asp Val Ser 230 235 240 ctg ccc cgt gga gcc ttt ccc ccc aga ggg ccc cgc cca gtg cag tcg 1003 Leu Pro Arg Gly Ala Phe Pro Pro Arg Gly Pro Arg Pro Val Gln Ser 245 250 255 gtg gta cct gag atg gag gag tcg gga gca cag ctg ctg ctg gaa atg 1051 Val Val Pro Glu Met Glu Glu Ser Gly Ala Gln Leu Leu Leu Glu Met 260 265 270 275 ctg act ttt aac cca cac aag cga atc tct gcc ttt cga gct ctg cag 1099 Leu Thr Phe Asn Pro His Lys Arg Ile Ser Ala Phe Arg Ala Leu Gln 280 285 290 cac tct tat cta cat aag gat gaa ggt aat ccg gag tga gcaatggagt 1148 His Ser Tyr Leu His Lys Asp Glu Gly Asn Pro Glu * 295 300 ggctgccatg gaaggaagaa aagctgccat ttcccttctg gacactgaga gggcaatctt 1208 tgcctttatc tctgaggcta tggagggtcc tcctccatct ttctacagag attactttgc 1268 tgccttaatg acattcccct cccacctctc cttttgaggc ttctccttct ccttcccatt 1328 tctctacact aaggggtatg ttccctcttg tccctttccc tacctttata tttggggtcc 1388 ttttttatac aggaaaaaca aaacaaagaa ataatggtct tttttttttt ttt 1441 7 303 PRT Mus musculus PEPTIDE (1)...(303) Balb/C mouse cdk4 7 Met Ala Ala Thr Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala 1 5 10 15 Tyr Gly Thr Val Tyr Lys Ala Arg Asp Pro His Ser Gly His Phe Val 20 25 30 Ala Leu Lys Ser Val Arg Val Pro Asn Gly Gly Ala Ala Gly Gly Gly 35 40 45 Leu Pro Val Ser Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu 50 55 60 Ala Phe Glu His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr 65 70 75 80 Ser Arg Thr Asp Arg Asp Ile Lys Val Thr Leu Val Phe Glu His Ile 85 90 95 Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys Ala Pro Pro Pro Gly Leu 100 105 110 Pro Val Glu Thr Ile Lys Asp Leu Met Arg Gln Phe Leu Ser Gly Leu 115 120 125 Asp Phe Leu His Ala Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu 130 135 140 Asn Ile Leu Val Thr Ser Asn Gly Thr Val Lys Leu Ala Asp Phe Gly 145 150 155 160 Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala Leu Thr Pro Val Val Val 165 170 175 Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala 180 185 190 Thr Pro Val Asp Met Trp Ser Val Gly Cys Ile Phe Ala Glu Met Phe 195 200 205 Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Gly 210 215 220 Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro Glu Asp Asp Trp Pro Arg 225 230 235 240 Glu Val Ser Leu Pro Arg Gly Ala Phe Ala Pro Arg Gly Pro Arg Pro 245 250 255 Val Gln Ser Val Val Pro Glu Met Glu Glu Ser Gly Ala Gln Leu Leu 260 265 270 Leu Glu Met Leu Thr Phe Asn Pro His Lys Arg Ile Ser Ala Phe Arg 275 280 285 Ala Leu Gln His Ser Tyr Leu His Lys Glu Glu Ser Asp Ala Glu 290 295 300 8 912 DNA Mus musculus mRNA (1)...(912) Balb/C mouse cdk4 cDNA 8 atg gct gcc act cga tat gaa ccc gtg gct gaa att ggt gtc ggt gcc 48 Met Ala Ala Thr Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala 1 5 10 15 tat ggg acg gtg tac aaa gcc cga gat ccc cac agt ggc cac ttt gtg 96 Tyr Gly Thr Val Tyr Lys Ala Arg Asp Pro His Ser Gly His Phe Val 20 25 30 gcc ctc aag agt gtg aga gtt cct aat gga gga gca gct gga ggg ggc 144 Ala Leu Lys Ser Val Arg Val Pro Asn Gly Gly Ala Ala Gly Gly Gly 35 40 45 ctt ccc gtc agc aca gtt cgt gag gtg gcc ttg tta agg agg ctg gag 192 Leu Pro Val Ser Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu 50 55 60 gcc ttt gaa cat ccc aat gtt gta cgg ctg atg gat gtc tgt gct act 240 Ala Phe Glu His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr 65 70 75 80 tcc cga act gat cgg gac atc aag gtc acc cta gtg ttt gag cat ata 288 Ser Arg Thr Asp Arg Asp Ile Lys Val Thr Leu Val Phe Glu His Ile 85 90 95 gac cag gac ctg agg aca tac ctg gac aaa gca cct cca ccg ggc ctg 336 Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys Ala Pro Pro Pro Gly Leu 100 105 110 ccg gtt gag acc att aag gat cta atg cgt cag ttt cta agc ggc ctg 384 Pro Val Glu Thr Ile Lys Asp Leu Met Arg Gln Phe Leu Ser Gly Leu 115 120 125 gat ttt ctt cat gca aac tgc att gtt cac cgg gac ctg aag cca gag 432 Asp Phe Leu His Ala Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu 130 135 140 aac att cta gtg aca agt aat ggg acc gtc aag ctg gct gac ttt ggc 480 Asn Ile Leu Val Thr Ser Asn Gly Thr Val Lys Leu Ala Asp Phe Gly 145 150 155 160 cta gct aga atc tac agc tac cag atg gcc ctc acg cct gtg gtg gtt 528 Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala Leu Thr Pro Val Val Val 165 170 175 acg ctc tgg tac cga gct cct gaa gtt ctt ctg cag tct aca tac gca 576 Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala 180 185 190 aca ccc gtg gac atg tgg agc gtt ggc tgt atc ttt gca gag atg ttc 624 Thr Pro Val Asp Met Trp Ser Val Gly Cys Ile Phe Ala Glu Met Phe 195 200 205 cgt cgg aag cct ctc ttc tgt gga aac tct gaa gcc gac cag ttg ggg 672 Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Gly 210 215 220 aaa atc ttt gat ctc att gga ttg cct cca gaa gac gac tgg cct cga 720 Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro Glu Asp Asp Trp Pro Arg 225 230 235 240 gag gta tct cta cct cga gga gcc ttt gcc ccc aga ggg cct cgg cca 768 Glu Val Ser Leu Pro Arg Gly Ala Phe Ala Pro Arg Gly Pro Arg Pro 245 250 255 gtg cag tca gtg gtg cca gag atg gag gag tct gga gcg cag ctg cta 816 Val Gln Ser Val Val Pro Glu Met Glu Glu Ser Gly Ala Gln Leu Leu 260 265 270 ctg gaa atg ctg acc ttt aac cca cat aag cga atc tct gcc ttc cga 864 Leu Glu Met Leu Thr Phe Asn Pro His Lys Arg Ile Ser Ala Phe Arg 275 280 285 gcc ctg cag cac tcc tac ctg cac aag gag gaa agc gac gca gag tga 912 Ala Leu Gln His Ser Tyr Leu His Lys Glu Glu Ser Asp Ala Glu * 290 295 300 9 303 PRT Rattus norvegicus PEPTIDE (1)...(303) rat cdk4 9 Met Ala Thr Thr Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala 1 5 10 15 Tyr Gly Thr Val Tyr Lys Ala Arg Asp Pro His Ser Gly His Phe Val 20 25 30 Ala Leu Lys Ser Val Arg Val Pro Asn Gly Gly Ala Ala Gly Gly Gly 35 40 45 Leu Pro Val Ser Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu 50 55 60 Ala Phe Glu His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr 65 70 75 80 Ser Arg Thr Asp Arg Asp Ile Lys Val Thr Leu Val Phe Glu His Ile 85 90 95 Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys Ala Pro Pro Pro Gly Leu 100 105 110 Pro Val Glu Thr Ile Lys Asp Leu Met Arg Gln Phe Leu Ser Gly Leu 115 120 125 Asp Phe Leu His Ala Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu 130 135 140 Asn Ile Leu Val Thr Ser Asn Gly Thr Val Lys Leu Ala Asp Phe Gly 145 150 155 160 Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala Leu Thr Pro Val Val Val 165 170 175 Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala 180 185 190 Thr Pro Val Asp Met Trp Ser Val Gly Cys Ile Phe Ala Glu Met Phe 195 200 205 Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Gly 210 215 220 Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro Glu Asp Asp Trp Pro Arg 225 230 235 240 Glu Val Ser Leu Pro Arg Gly Ala Phe Ser Pro Arg Gly Pro Arg Pro 245 250 255 Val Gln Ser Val Val Pro Glu Met Glu Glu Ser Gly Ala Gln Leu Leu 260 265 270 Leu Glu Met Leu Thr Phe Asn Pro Leu Lys Arg Ile Ser Ala Phe Arg 275 280 285 Ala Leu Gln His Ser Tyr Leu His Lys Glu Glu Ser Asp Pro Glu 290 295 300 10 1232 DNA Rattus norvegicus mRNA (1)...(1232) rat cdk4 cDNA 10 ccgaagcgtc cagctgcccg ggaccgatcc ccggtgtatg gcgccgcaga aacggctccc 60 gggcccagat aaagggcacc tccgcagctc ttggccgaga gatcccctgc tccgaga atg 120 Met 1 gct acc act cga tat gaa ccc gtg gct gaa att ggt gtc ggt gcc tat 168 Ala Thr Thr Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala Tyr 5 10 15 ggg acg gtg tac aaa gcc cga gat ccc cac agt ggc cac ttt gtg gct 216 Gly Thr Val Tyr Lys Ala Arg Asp Pro His Ser Gly His Phe Val Ala 20 25 30 ctc aag agt gtg aga gtt cct aat gga gga gca gct gga ggg ggc ctt 264 Leu Lys Ser Val Arg Val Pro Asn Gly Gly Ala Ala Gly Gly Gly Leu 35 40 45 ccc gtc agc aca gtt cgt gag gtg gcc ttg tta aga agg ctg gag gcc 312 Pro Val Ser Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu Ala 50 55 60 65 ttt gaa cat ccc aat gtt gta cgg ctg atg gat gtc tgt gct act tcc 360 Phe Glu His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr Ser 70 75 80 cga act gat cgg gac atc aag gtc acc tta gtg ttt gag cat ata gac 408 Arg Thr Asp Arg Asp Ile Lys Val Thr Leu Val Phe Glu His Ile Asp 85 90 95 cag gac cta cgg aca tac ctg gac aaa gca cct ccg ccg ggc ttg cct 456 Gln Asp Leu Arg Thr Tyr Leu Asp Lys Ala Pro Pro Pro Gly Leu Pro 100 105 110 gtt gag acc att aag gat ctg atg cgc cag ttt cta agc ggc cta gat 504 Val Glu Thr Ile Lys Asp Leu Met Arg Gln Phe Leu Ser Gly Leu Asp 115 120 125 ttc ctt cat gca aac tgc att gtt cac cgg gac ctg aag cca gag aac 552 Phe Leu His Ala Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu Asn 130 135 140 145 att cta gtg aca agt aat ggg aca gtt aag ctg gcc gac ttt ggc cta 600 Ile Leu Val Thr Ser Asn Gly Thr Val Lys Leu Ala Asp Phe Gly Leu 150 155 160 gcc aga atc tac agc tac cag atg gcc ctc acg cct gtg gtt gtt acg 648 Ala Arg Ile Tyr Ser Tyr Gln Met Ala Leu Thr Pro Val Val Val Thr 165 170 175 ctc tgg tac cgg gct cct gaa gtt ctt ctg cag tct aca tat gca acg 696 Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala Thr 180 185 190 cct gtg gat atg tgg agt gtt ggc tgt atc ttc gca gag atg ttt cgc 744 Pro Val Asp Met Trp Ser Val Gly Cys Ile Phe Ala Glu Met Phe Arg 195 200 205 cgg aag cct ctc ttc tgt ggg aac tct gag gct gac cag ctg ggc aaa 792 Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Gly Lys 210 215 220 225 atc ttt gat ctc att gga ttg cct cca gaa gac gac tgg cct cga gag 840 Ile Phe Asp Leu Ile Gly Leu Pro Pro Glu Asp Asp Trp Pro Arg Glu 230 235 240 gtc tct ctt cct cga gga gcc ttt tcc ccc aga gga cct cgg cca gtg 888 Val Ser Leu Pro Arg Gly Ala Phe Ser Pro Arg Gly Pro Arg Pro Val 245 250 255 cag tca gtg gtg ccg gag atg gag gaa tct gga gcg cag ttg ctg ctg 936 Gln Ser Val Val Pro Glu Met Glu Glu Ser Gly Ala Gln Leu Leu Leu 260 265 270 gaa atg ctg acc ttt aat cca ctt aag cga atc tct gcc ttc cga gcc 984 Glu Met Leu Thr Phe Asn Pro Leu Lys Arg Ile Ser Ala Phe Arg Ala 275 280 285 ctg cag cac tct tac ctg cac aag gag gaa agt gac ccg gag tga 1029 Leu Gln His Ser Tyr Leu His Lys Glu Glu Ser Asp Pro Glu * 290 295 300 ggagaggggc tgccttttcc agtttccgga cgctgtgtgg aaagaacctt gctgaagtgg 1089 cagcctctgc cttcccctga ggctatggag agtcctccgg tttagcctta aatgacaatt 1149 ccctacctct ccttatgagg tttaccccac ccccaccctc ctgtttctct acactaaagg 1209 gcaggcatta tctgtcttct tcc 1232 11 303 PRT Sus scrofa 11 Met Ala Thr Ser Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala 1 5 10 15 Tyr Gly Thr Val Tyr Lys Ala Arg Asp Pro His Ser Gly His Phe Val 20 25 30 Ala Leu Lys Ser Val Arg Val Pro Asn Gly Gly Gly Ala Gly Gly Gly 35 40 45 Leu Pro Ile Ser Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu 50 55 60 Ala Phe Glu His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr 65 70 75 80 Ala Arg Thr Asp Arg Glu Thr Lys Val Thr Leu Val Phe Glu His Val 85 90 95 Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys Ala Pro Pro Pro Gly Leu 100 105 110 Pro Val Glu Thr Ile Lys Asp Leu Met Arg Gln Phe Leu Arg Gly Leu 115 120 125 Asp Phe Leu His Ala Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu 130 135 140 Asn Ile Leu Val Thr Ser Gly Gly Thr Val Lys Leu Ala Asp Phe Gly 145 150 155 160 Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala Leu Thr Pro Val Val Val 165 170 175 Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala 180 185 190 Thr Pro Val Asp Met Trp Ser Val Gly Cys Ile Phe Ala Glu Met Phe 195 200 205 Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Gly 210 215 220 Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro Glu Asp Asp Trp Pro Arg 225 230 235 240 Asp Val Ser Leu Pro Arg Gly Ala Phe Ser Pro Arg Gly Pro Arg Pro 245 250 255 Val Gln Ser Val Val Pro Glu Met Glu Glu Ser Gly Ala Gln Leu Leu 260 265 270 Leu Glu Met Leu Thr Phe Asn Pro His Lys Arg Ile Ser Ala Phe Arg 275 280 285 Ala Leu Gln His Ser Tyr Leu His Lys Ala Glu Gly Asn Pro Glu 290 295 300 12 912 DNA Sus scrofa CDS (1)...(912) domestic pig cdk4 coding sequence 12 atg gct acc tcc cgg tat gaa cca gtg gcg gag att ggt gtt ggt gcc 48 Met Ala Thr Ser Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala 1 5 10 15 tat ggg acc gtg tac aaa gca cgg gat ccc cac agt ggc cac ttt gtg 96 Tyr Gly Thr Val Tyr Lys Ala Arg Asp Pro His Ser Gly His Phe Val 20 25 30 gcc ctc aag agc gta aga gtc ccc aat gga gga ggt gct gga ggg ggc 144 Ala Leu Lys Ser Val Arg Val Pro Asn Gly Gly Gly Ala Gly Gly Gly 35 40 45 ctg ccc atc agc acg gtt cgt gaa gtg gcc tta ctg cgc cgg ctg gag 192 Leu Pro Ile Ser Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu 50 55 60 gct ttc gag cat ccc aat gtt gtc cgg ctg atg gat gtc tgt gcc act 240 Ala Phe Glu His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr 65 70 75 80 gcc cga act gat cga gag acc aaa gtg acc ctg gtg ttt gag cat gtg 288 Ala Arg Thr Asp Arg Glu Thr Lys Val Thr Leu Val Phe Glu His Val 85 90 95 gac caa gat cta agg aca tat ctg gac aag gca ccc cca cca ggc ttg 336 Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys Ala Pro Pro Pro Gly Leu 100 105 110 ccg gtg gag acc atc aag gat ctg atg cgt cag ttt cta aga ggc cta 384 Pro Val Glu Thr Ile Lys Asp Leu Met Arg Gln Phe Leu Arg Gly Leu 115 120 125 gat ttc ctt cat gcc aac tgc atc gtt cat cga gac ctg aag cca gag 432 Asp Phe Leu His Ala Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu 130 135 140 aac att ctg gtt aca agt ggt ggg aca gtc aag ctg gct gac ttt ggc 480 Asn Ile Leu Val Thr Ser Gly Gly Thr Val Lys Leu Ala Asp Phe Gly 145 150 155 160 ctg gcc aga atc tac agc tac cag atg gca ctt aca cct gtg gtg gtt 528 Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala Leu Thr Pro Val Val Val 165 170 175 aca ctc tgg tac cgt gct cca gaa gtt ctt ttg cag tct acg tat gca 576 Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala 180 185 190 aca cct gtg gac atg tgg agc gtt ggc tgt atc ttc gca gag atg ttt 624 Thr Pro Val Asp Met Trp Ser Val Gly Cys Ile Phe Ala Glu Met Phe 195 200 205 cgt cga aag cct ctc ttc tgt gga aac tct gaa gct gac cag tta ggc 672 Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Gly 210 215 220 aaa atc ttt gac ctg att ggg ctg ccc cca gag gat gac tgg ccc cga 720 Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro Glu Asp Asp Trp Pro Arg 225 230 235 240 gat gtg tct cta ccc cga gga gcc ttt tcc ccc aga ggg ccc cgc cca 768 Asp Val Ser Leu Pro Arg Gly Ala Phe Ser Pro Arg Gly Pro Arg Pro 245 250 255 gtg cag tcg gtg gta cct gag atg gag gag tct gga gca cag ctg cta 816 Val Gln Ser Val Val Pro Glu Met Glu Glu Ser Gly Ala Gln Leu Leu 260 265 270 ctg gag atg ctg act ttt aac cca cac aag cga atc tct gcc ttc cga 864 Leu Glu Met Leu Thr Phe Asn Pro His Lys Arg Ile Ser Ala Phe Arg 275 280 285 gcc ctg cag cac tct tat cta cat aag gca gaa ggt aac cca gag tga 912 Ala Leu Gln His Ser Tyr Leu His Lys Ala Glu Gly Asn Pro Glu * 290 295 300 13 319 PRT Xenopus laevis 13 Met Ser Lys Glu Met Lys Gly Gln Tyr Glu Pro Val Ala Glu Ile Gly 1 5 10 15 Val Gly Ala Tyr Gly Thr Val Tyr Lys Ala Arg Asp Leu Gln Ser Gly 20 25 30 Lys Phe Val Ala Leu Lys Asn Val Arg Val Gln Thr Asn Glu Asn Gly 35 40 45 Leu Pro Leu Ser Thr Val Arg Glu Val Thr Leu Leu Lys Arg Leu Glu 50 55 60 His Phe Asp His Pro Asn Ile Val Lys Leu Met Asp Val Cys Ala Ser 65 70 75 80 Ala Arg Thr Asp Arg Glu Thr Lys Val Thr Leu Val Phe Glu His Val 85 90 95 Asp Gln Asp Leu Lys Thr Tyr Leu Ser Lys Val Pro Pro Pro Gly Leu 100 105 110 Pro Leu Glu Thr Ile Lys Asp Leu Met Lys Gln Phe Leu Ser Gly Leu 115 120 125 Glu Phe Leu His Leu Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu 130 135 140 Asn Ile Leu Val Thr Ser Gly Gly Gln Val Lys Leu Ala Asp Phe Gly 145 150 155 160 Leu Ala Arg Ile Tyr Ser Cys Gln Met Ala Leu Thr Pro Val Val Val 165 170 175 Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala 180 185 190 Thr Pro Val Asp Val Trp Ser Ala Gly Cys Ile Phe Ala Glu Met Phe 195 200 205 Lys Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Cys 210 215 220 Lys Ile Phe Asp Ile Ile Gly Leu Pro Ser Glu Glu Glu Trp Pro Val 225 230 235 240 Asp Val Thr Leu Pro Arg Ser Ala Phe Ser Pro Arg Thr Gln Gln Pro 245 250 255 Val Asp Lys Phe Val Pro Glu Ile Asp Ala Met Gly Ala Asp Leu Leu 260 265 270 Leu Ala Met Leu Thr Phe Ser Pro Gln Lys Arg Ile Ser Ala Ser Asp 275 280 285 Ala Leu Leu His Pro Phe Phe Ala Asp Asp Pro Gln Ala Cys Ser Lys 290 295 300 Gln Glu His Phe Thr His Ile Cys Thr Ala Thr Asp Glu Val Lys 305 310 315 14 2258 DNA Xenopus laevis mRNA (1)...(2258) X. laevis cDNA 14 ggcacgagcg gcacgagcat cctcctcatg ctagcgtgca gcagtgcaag gcatggcgtg 60 aatgcgggcg cttccatgtc aggagatcag cgctgtgagg ttcccgtgct attcaggaca 120 gggggttctt gagaagaata ccagtgtgga at atg tca aaa gaa atg aag ggt 173 Met Ser Lys Glu Met Lys Gly 1 5 caa tat gaa cct gtc gca gag att ggc gtc ggg gct tac ggc acg gta 221 Gln Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala Tyr Gly Thr Val 10 15 20 tac aag gcc agg gac ctt caa agt gga aag ttt gtt gcc ctc aaa aat 269 Tyr Lys Ala Arg Asp Leu Gln Ser Gly Lys Phe Val Ala Leu Lys Asn 25 30 35 gtg cgg gtg caa acc aac gag aat gga ctc ccc ctc tct acg gtc agg 317 Val Arg Val Gln Thr Asn Glu Asn Gly Leu Pro Leu Ser Thr Val Arg 40 45 50 55 gaa gtg act tta ttg aaa cgc ctg gag cat ttt gac cat cca aac att 365 Glu Val Thr Leu Leu Lys Arg Leu Glu His Phe Asp His Pro Asn Ile 60 65 70 gta aaa ctt atg gat gtt tgt gct tct gcc cga act gac cga gag act 413 Val Lys Leu Met Asp Val Cys Ala Ser Ala Arg Thr Asp Arg Glu Thr 75 80 85 aaa gtg acg ctg gtg ttt gaa cat gtg gac caa gac ctg aag act tac 461 Lys Val Thr Leu Val Phe Glu His Val Asp Gln Asp Leu Lys Thr Tyr 90 95 100 ttg agc aaa gtc cca cct cca ggt ctt cca ctg gag act att aag gac 509 Leu Ser Lys Val Pro Pro Pro Gly Leu Pro Leu Glu Thr Ile Lys Asp 105 110 115 ctc atg aag cag ttc ctt tcg ggc ctg gaa ttc ctc cat ttg aat tgc 557 Leu Met Lys Gln Phe Leu Ser Gly Leu Glu Phe Leu His Leu Asn Cys 120 125 130 135 att gtg cac cga gac ctt aaa ccg gag aat atc ttg gtg aca agt ggg 605 Ile Val His Arg Asp Leu Lys Pro Glu Asn Ile Leu Val Thr Ser Gly 140 145 150 gga cag gtg aaa ctt gcc gac ttt ggc cta gcg agg ata tac agc tgt 653 Gly Gln Val Lys Leu Ala Asp Phe Gly Leu Ala Arg Ile Tyr Ser Cys 155 160 165 cag atg gct ctc acg ccc gtg gtg gtg acg ttg tgg tac agg gcc cca 701 Gln Met Ala Leu Thr Pro Val Val Val Thr Leu Trp Tyr Arg Ala Pro 170 175 180 gaa gtc ctg ctt cag tcc acg tat gcc acg ccg gta gat gtg tgg agc 749 Glu Val Leu Leu Gln Ser Thr Tyr Ala Thr Pro Val Asp Val Trp Ser 185 190 195 gcg ggc tgc atc ttc gca gag atg ttc aag cga aaa ccc ctg ttc tgc 797 Ala Gly Cys Ile Phe Ala Glu Met Phe Lys Arg Lys Pro Leu Phe Cys 200 205 210 215 ggc aat tca gaa gct gac cag ttg tgc aag ata ttt gat att att ggg 845 Gly Asn Ser Glu Ala Asp Gln Leu Cys Lys Ile Phe Asp Ile Ile Gly 220 225 230 ctt cct tct gaa gag gag tgg ccg gtg gat gtt act ttg cca cgt tct 893 Leu Pro Ser Glu Glu Glu Trp Pro Val Asp Val Thr Leu Pro Arg Ser 235 240 245 gcc ttt tcc ccg agg act cag cag cca gtg gac aag ttt gtg ccg gaa 941 Ala Phe Ser Pro Arg Thr Gln Gln Pro Val Asp Lys Phe Val Pro Glu 250 255 260 atc gat gcc atg gga gcc gat ctt ctt ctg gcc atg ctg acc ttc agc 989 Ile Asp Ala Met Gly Ala Asp Leu Leu Leu Ala Met Leu Thr Phe Ser 265 270 275 cct caa aaa cgt att tcc gcc tcc gat gct ctg ctc cac ccc ttt ttc 1037 Pro Gln Lys Arg Ile Ser Ala Ser Asp Ala Leu Leu His Pro Phe Phe 280 285 290 295 gct gac gat ccc cag gcc tgc tcc aaa caa gaa cac ttt act cat atc 1085 Ala Asp Asp Pro Gln Ala Cys Ser Lys Gln Glu His Phe Thr His Ile 300 305 310 tgc acc gca acg gat gaa gtg aaa tga aacggcatcc ctgcctgggg 1132 Cys Thr Ala Thr Asp Glu Val Lys * 315 gctccagggt cggcgcagat gtgggcacat tatttgcttt ttctctgcct taaagggagt 1192 tctcagcggg aggggaaagc aaataagcga taaagtgacc atgtgatcct tgtaaaacat 1252 ttccacttcc tggttgtaat ggattttatg ctgcaaacgt cactgtgacc gacgcaagat 1312 tccgctgttc agatcagcgc cgtttcgcat ttgtgcctta tcggtcctta catttatggt 1372 ttggggagct cccttaacca gagacgcaca ataagctaca ctatcctcat tatttctgtt 1432 ctcaccgcat tgatggtgtc aaatctcggc tttaccacaa tttttatcac ggtgctcttt 1492 ctggggtaaa aaaacaaaaa agacttattg atcatgtgct aaaatttagc gcacgccaga 1552 ctgttcgaga gcccgacaaa taatttctac catttttgta ctgttttaca ataatgtttt 1612 atattggatt ttcaagggta tttctattgc gctggggtgg ggtaaaaatt gacctgcggc 1672 tattttgttc ctatggtaaa aacatggcag tcttaaccca ctagggatcc acggaatccg 1732 ctattttttt tgttaggctg aatcctctac aaaagattct gcaaaatctg aacccaaatt 1792 tgcaagttca tattggataa aaaccaagca tttcacattc agttattgag agcttaaaag 1852 acaaatggcc atgagggtta ggattcggcc gaatgttaat gtttcattaa agtgctaggt 1912 tttaaccaaa gcaacaacca aaacttggcc ttgttgcatc cctacaactg acatcttgat 1972 ctttcatgct ggtgaatgtt ggacggtcat gggcccattg tgggtataat ttgttcaatt 2032 tggagcctgc ctcgctaaat cctttaatgc agaattcttt ccttttctga actgcccttg 2092 tttgtctgtt gacattgaaa ccccattatc gtcgttggtt caactgcgca cgctgtcgtt 2152 ctgaatgtta cagttcagct ttaaagtcta ctcataaagt gaagtttaaa gcgggtggga 2212 attgccagat ttagtgtgag ggtgaaaaaa aaaaaaaact cgtgcc 2258 15 303 PRT Homo sapiens 15 Met Ala Thr Ser Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala 1 5 10 15 Tyr Gly Thr Val Tyr Lys Ala Cys Asp Pro His Ser Gly His Phe Val 20 25 30 Ala Leu Lys Ser Val Arg Val Pro Asn Gly Gly Gly Gly Gly Gly Gly 35 40 45 Leu Pro Ile Ser Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu 50 55 60 Ala Phe Glu His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr 65 70 75 80 Ser Arg Thr Asp Arg Glu Ile Lys Val Thr Leu Val Phe Glu His Val 85 90 95 Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys Ala Pro Pro Pro Gly Leu 100 105 110 Pro Ala Glu Thr Ile Lys Asp Leu Met Arg Gln Phe Leu Arg Gly Leu 115 120 125 Asp Phe Leu His Ala Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu 130 135 140 Asn Ile Leu Val Thr Ser Gly Gly Thr Val Lys Leu Ala Asp Phe Gly 145 150 155 160 Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala Leu Thr Pro Val Val Val 165 170 175 Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala 180 185 190 Thr Pro Val Asp Met Trp Ser Val Gly Cys Ile Phe Ala Glu Met Phe 195 200 205 Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Gly 210 215 220 Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro Glu Asp Asp Trp Pro Arg 225 230 235 240 Asp Val Ser Leu Pro Arg Gly Ala Phe Pro Pro Arg Gly Pro Arg Pro 245 250 255 Val Gln Ser Val Val Pro Glu Met Glu Glu Ser Gly Ala Gln Leu Leu 260 265 270 Leu Glu Met Leu Thr Phe Asn Pro His Lys Arg Ile Ser Ala Phe Arg 275 280 285 Ala Leu Gln His Ser Tyr Leu His Lys Asp Glu Gly Asn Pro Glu 290 295 300 16 1351 DNA Homo sapiens mRNA (1)...(1351) Human cdk4R24C mutant 16 gtcgggccct ctgcgtccag ctgctccgga ccgagctcgg gtgtatgggg ccgtaggaac 60 cggctccggg gccccgataa cgggccgccc ccacagcacc ccgggctggc gtgagggtct 120 cccttgatct gaga atg gct acc tct cga tat gag cca gtg gct gaa att 170 Met Ala Thr Ser Arg Tyr Glu Pro Val Ala Glu Ile 1 5 10 ggt gtc ggt gcc tat ggg aca gtg tac aag gcc tgt gat ccc cac agt 218 Gly Val Gly Ala Tyr Gly Thr Val Tyr Lys Ala Cys Asp Pro His Ser 15 20 25 ggc cac ttt gtg gcc ctc aag agt gtg aga gtc ccc aat gga gga gga 266 Gly His Phe Val Ala Leu Lys Ser Val Arg Val Pro Asn Gly Gly Gly 30 35 40 ggt gga gga ggc ctt ccc atc agc aca gtt cgt gag gtg gct tta ctg 314 Gly Gly Gly Gly Leu Pro Ile Ser Thr Val Arg Glu Val Ala Leu Leu 45 50 55 60 agg cga ctg gag gct ttt gag cat ccc aat gtt gtc cgg ctg atg gac 362 Arg Arg Leu Glu Ala Phe Glu His Pro Asn Val Val Arg Leu Met Asp 65 70 75 gtc tgt gcc aca tcc cga act gac cgg gag atc aag gta acc ctg gtg 410 Val Cys Ala Thr Ser Arg Thr Asp Arg Glu Ile Lys Val Thr Leu Val 80 85 90 ttt gag cat gta gac cag gac cta agg aca tat ctg gac aag gca ccc 458 Phe Glu His Val Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys Ala Pro 95 100 105 cca cca ggc ttg cca gcc gaa acg atc aag gat ctg atg cgc cag ttt 506 Pro Pro Gly Leu Pro Ala Glu Thr Ile Lys Asp Leu Met Arg Gln Phe 110 115 120 cta aga ggc cta gat ttc ctt cat gcc aat tgc atc gtt cac cga gat 554 Leu Arg Gly Leu Asp Phe Leu His Ala Asn Cys Ile Val His Arg Asp 125 130 135 140 ctg aag cca gag aac att ctg gtg aca agt ggt gga aca gtc aag ctg 602 Leu Lys Pro Glu Asn Ile Leu Val Thr Ser Gly Gly Thr Val Lys Leu 145 150 155 gct gac ttt ggc ctg gcc aga atc tac agc tac cag atg gca ctt aca 650 Ala Asp Phe Gly Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala Leu Thr 160 165 170 ccc gtg gtt gtt aca ctc tgg tac cga gct ccc gaa gtt ctt ctg cag 698 Pro Val Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln 175 180 185 tcc aca tat gca aca cct gtg gac atg tgg agt gtt ggc tgt atc ttt 746 Ser Thr Tyr Ala Thr Pro Val Asp Met Trp Ser Val Gly Cys Ile Phe 190 195 200 gca gag atg ttt cgt cga aag cct ctc ttc tgt gga aac tct gaa gcc 794 Ala Glu Met Phe Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala 205 210 215 220 gac cag ttg ggc aaa atc ttt gac ctg att ggg ctg cct cca gag gat 842 Asp Gln Leu Gly Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro Glu Asp 225 230 235 gac tgg cct cga gat gta tcc ctg ccc cgt gga gcc ttt ccc ccc aga 890 Asp Trp Pro Arg Asp Val Ser Leu Pro Arg Gly Ala Phe Pro Pro Arg 240 245 250 ggg ccc cgc cca gtg cag tcg gtg gta cct gag atg gag gag tcg gga 938 Gly Pro Arg Pro Val Gln Ser Val Val Pro Glu Met Glu Glu Ser Gly 255 260 265 gca cag ctg ctg ctg gaa atg ctg act ttt aac cca cac aag cga atc 986 Ala Gln Leu Leu Leu Glu Met Leu Thr Phe Asn Pro His Lys Arg Ile 270 275 280 tct gcc ttt cga gct ctg cag cac tct tat cta cat aag gat gaa ggt 1034 Ser Ala Phe Arg Ala Leu Gln His Ser Tyr Leu His Lys Asp Glu Gly 285 290 295 300 aat ccg gag tga gcaatggagt ggctgccatg gaaggaagaa aagctgccat 1086 Asn Pro Glu * ttcccttctg gacactgaga gggcaatctt tgcctttatc tctgaggcta tggagggtcc 1146 tcctccatct ttctacagag attactttgc tgccttaatg acattcccct cccacctctc 1206 cttttgaggc ttctccttct ccttcccatt tctctacact aaggggtatg ttccctcttg 1266 tccctttccc tacctttata tttggggtcc ttttttatac aggaaaaaca aaacaaagaa 1326 aaaaagtcga cgcggccgcg aattc 1351 17 326 PRT Homo sapiens 17 Met Glu Lys Asp Gly Leu Cys Arg Ala Asp Gln Gln Tyr Glu Cys Val 1 5 10 15 Ala Glu Ile Gly Glu Gly Ala Tyr Gly Lys Val Phe Lys Ala Arg Asp 20 25 30 Leu Lys Asn Gly Gly Arg Phe Val Ala Leu Lys Arg Val Arg Val Gln 35 40 45 Thr Gly Glu Glu Gly Met Pro Leu Ser Thr Ile Arg Glu Val Ala Val 50 55 60 Leu Arg His Leu Glu Thr Phe Glu His Pro Asn Val Val Arg Leu Phe 65 70 75 80 Asp Val Cys Thr Val Ser Arg Thr Asp Arg Glu Thr Lys Leu Thr Leu 85 90 95 Val Phe Glu His Val Asp Gln Asp Leu Thr Thr Tyr Leu Asp Lys Val 100 105 110 Pro Glu Pro Gly Val Pro Thr Glu Thr Ile Lys Asp Met Met Phe Gln 115 120 125 Leu Leu Arg Gly Leu Asp Phe Leu His Ser His Arg Val Val His Arg 130 135 140 Asp Leu Lys Pro Gln Asn Ile Leu Val Thr Ser Ser Gly Gln Ile Lys 145 150 155 160 Leu Ala Asp Phe Gly Leu Ala Arg Ile Tyr Ser Phe Gln Met Ala Leu 165 170 175 Thr Ser Val Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu 180 185 190 Gln Ser Ser Tyr Ala Thr Pro Val Asp Leu Trp Ser Val Gly Cys Ile 195 200 205 Phe Ala Glu Met Phe Arg Arg Lys Pro Leu Phe Arg Gly Ser Ser Asp 210 215 220 Val Asp Gln Leu Gly Lys Ile Leu Asp Val Ile Gly Leu Pro Gly Glu 225 230 235 240 Glu Asp Trp Pro Arg Asp Val Ala Leu Pro Arg Gln Ala Phe His Ser 245 250 255 Lys Ser Ala Gln Pro Ile Glu Lys Phe Val Thr Asp Ile Asp Glu Leu 260 265 270 Gly Lys Asp Leu Leu Leu Lys Cys Leu Thr Phe Asn Pro Ala Lys Arg 275 280 285 Ile Ser Ala Tyr Ser Ala Leu Ser His Pro Tyr Phe Gln Asp Leu Glu 290 295 300 Arg Cys Lys Glu Asn Leu Asp Ser His Leu Pro Pro Ser Gln Asn Thr 305 310 315 320 Ser Glu Leu Asn Thr Ala 325 18 1247 DNA Homo sapiens 18 gtaaagctag accgatctcc ggggagcccc ggagtaggcg agcggcggcc gccagctagt 60 tgagcgcacc ccccgcccgc cccagcggcg ccgcggcggg cggcgtccag gcggcatgga 120 gaaggacggc ctgtgccgcg ctgaccagca gtacgaatgc gtggcggaga tcggggaggg 180 cgcctatggg aaggtgttca aggcccgcga cttgaagaac ggaggccgtt tcgtggcgtt 240 gaagcgcgtg cgggtgcaga ccggcgagga gggcatgccg ctctccacca tccgcgaggt 300 ggcggtgctg aggcacctgg agaccttcga gcaccccaac gtggtcaggt tgtttgatgt 360 gtgcacagtg tcacgaacag acagagaaac caaactaact ttagtgtttg aacatgtcga 420 tcaagacttg accacttact tggataaagt tccagagcct ggagtgccca ctgaaaccat 480 aaaggatatg atgtttcagc ttctccgagg tctggacttt cttcattcac accgagtagt 540 gcatcgcgat ctaaaaccac agaacattct ggtgaccagc agcggacaaa taaaactcgc 600 tgacttcggc cttgcccgca tctatagttt ccagatggct ctaacctcag tggtcgtcac 660 gctgtggtac agagcacccg aagtcttgct ccagtccagc tacgccaccc ccgtggatct 720 ctggagtgtt ggctgcatat ttgcagaaat gtttcgtaga aagcctcttt ttcgtggaag 780 ttcagatgtt gatcaactag gaaaaatctt ggacgtgatt ggactcccag gagaagaaga 840 ctggcctaga gatgttgccc ttcccaggca ggcttttcat tcaaaatctg cccaaccaat 900 tgagaagttt gtaacagata tcgatgaact aggcaaagac ctacttctga agtgtttgac 960 atttaaccca gccaaaagaa tatctgccta cagtgccctg tctcacccat acttccagga 1020 cctggaaagg tgcaaagaaa acctggattc ccacctgccg cccagccaga acacctcgga 1080 gctgaataca gcctgaggcc tcagcagccg ccttaagctg atcctgcgga gaacaccctt 1140 ggtggcttat gggtccccct cagcaagccc tacagagctg tggaggattg ctatctggag 1200 gccttccagc tgctgtcttc tggacaggct ctgcttctcc aaggaaa 1247 19 326 PRT Homo sapiens 19 Met Glu Lys Asp Gly Leu Cys Arg Ala Asp Gln Gln Tyr Glu Cys Val 1 5 10 15 Ala Glu Ile Gly Glu Gly Ala Tyr Gly Lys Val Phe Lys Ala Cys Asp 20 25 30 Leu Lys Asn Gly Gly Arg Phe Val Ala Leu Lys Arg Val Arg Val Gln 35 40 45 Thr Gly Glu Glu Gly Met Pro Leu Ser Thr Ile Arg Glu Val Ala Val 50 55 60 Leu Arg His Leu Glu Thr Phe Glu His Pro Asn Val Val Arg Leu Phe 65 70 75 80 Asp Val Cys Thr Val Ser Arg Thr Asp Arg Glu Thr Lys Leu Thr Leu 85 90 95 Val Phe Glu His Val Asp Gln Asp Leu Thr Thr Tyr Leu Asp Lys Val 100 105 110 Pro Glu Pro Gly Val Pro Thr Glu Thr Ile Lys Asp Met Met Phe Gln 115 120 125 Leu Leu Arg Gly Leu Asp Phe Leu His Ser His Arg Val Val His Arg 130 135 140 Asp Leu Lys Pro Gln Asn Ile Leu Val Thr Ser Ser Gly Gln Ile Lys 145 150 155 160 Leu Ala Asp Phe Gly Leu Ala Arg Ile Tyr Ser Phe Gln Met Ala Leu 165 170 175 Thr Ser Val Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu 180 185 190 Gln Ser Ser Tyr Ala Thr Pro Val Asp Leu Trp Ser Val Gly Cys Ile 195 200 205 Phe Ala Glu Met Phe Arg Arg Lys Pro Leu Phe Arg Gly Ser Ser Asp 210 215 220 Val Asp Gln Leu Gly Lys Ile Leu Asp Val Ile Gly Leu Pro Gly Glu 225 230 235 240 Glu Asp Trp Pro Arg Asp Val Ala Leu Pro Arg Gln Ala Phe His Ser 245 250 255 Lys Ser Ala Gln Pro Ile Glu Lys Phe Val Thr Asp Ile Asp Glu Leu 260 265 270 Gly Lys Asp Leu Leu Leu Lys Cys Leu Thr Phe Asn Pro Ala Lys Arg 275 280 285 Ile Ser Ala Tyr Ser Ala Leu Ser His Pro Tyr Phe Gln Asp Leu Glu 290 295 300 Arg Cys Lys Glu Asn Leu Asp Ser His Leu Pro Pro Ser Gln Asn Thr 305 310 315 320 Ser Glu Leu Asn Thr Ala 325 20 1247 DNA Homo sapiens 20 gtaaagctag accgatctcc ggggagcccc ggagtaggcg agcggcggcc gccagctagt 60 tgagcgcacc ccccgcccgc cccagcggcg ccgcggcggg cggcgtccag gcggcatgga 120 gaaggacggc ctgtgccgcg ctgaccagca gtacgaatgc gtggcggaga tcggggaggg 180 cgcctatggg aaggtgttca aggcctgcga cttgaagaac ggaggccgtt tcgtggcgtt 240 gaagcgcgtg cgggtgcaga ccggcgagga gggcatgccg ctctccacca tccgcgaggt 300 ggcggtgctg aggcacctgg agaccttcga gcaccccaac gtggtcaggt tgtttgatgt 360 gtgcacagtg tcacgaacag acagagaaac caaactaact ttagtgtttg aacatgtcga 420 tcaagacttg accacttact tggataaagt tccagagcct ggagtgccca ctgaaaccat 480 aaaggatatg atgtttcagc ttctccgagg tctggacttt cttcattcac accgagtagt 540 gcatcgcgat ctaaaaccac agaacattct ggtgaccagc agcggacaaa taaaactcgc 600 tgacttcggc cttgcccgca tctatagttt ccagatggct ctaacctcag tggtcgtcac 660 gctgtggtac agagcacccg aagtcttgct ccagtccagc tacgccaccc ccgtggatct 720 ctggagtgtt ggctgcatat ttgcagaaat gtttcgtaga aagcctcttt ttcgtggaag 780 ttcagatgtt gatcaactag gaaaaatctt ggacgtgatt ggactcccag gagaagaaga 840 ctggcctaga gatgttgccc ttcccaggca ggcttttcat tcaaaatctg cccaaccaat 900 tgagaagttt gtaacagata tcgatgaact aggcaaagac ctacttctga agtgtttgac 960 atttaaccca gccaaaagaa tatctgccta cagtgccctg tctcacccat acttccagga 1020 cctggaaagg tgcaaagaaa acctggattc ccacctgccg cccagccaga acacctcgga 1080 gctgaataca gcctgaggcc tcagcagccg ccttaagctg atcctgcgga gaacaccctt 1140 ggtggcttat gggtccccct cagcaagccc tacagagctg tggaggattg ctatctggag 1200 gccttccagc tgctgtcttc tggacaggct ctgcttctcc aaggaaa 1247 21 326 PRT Mus musculus (Balb/c) 21 Met Glu Lys Asp Ser Leu Ser Arg Ala Asp Gln Gln Tyr Glu Cys Val 1 5 10 15 Ala Glu Ile Gly Glu Gly Ala Tyr Gly Lys Val Phe Lys Ala Arg Asp 20 25 30 Leu Lys Asn Gly Gly Arg Phe Val Ala Leu Lys Arg Val Arg Val Gln 35 40 45 Thr Ser Glu Glu Gly Met Pro Leu Ser Thr Ile Arg Glu Val Ala Val 50 55 60 Leu Arg His Leu Glu Thr Phe Glu His Pro Asn Val Val Arg Leu Phe 65 70 75 80 Asp Val Cys Thr Val Ser Arg Thr Asp Arg Glu Thr Lys Leu Thr Leu 85 90 95 Val Phe Glu His Val Asp Gln Asp Leu Thr Thr Tyr Leu Asp Lys Val 100 105 110 Pro Glu Pro Gly Val Pro Thr Glu Thr Ile Lys Asp Met Met Phe Gln 115 120 125 Leu Leu Arg Gly Leu Asp Phe Leu His Ser His Arg Val Val His Arg 130 135 140 Asp Leu Lys Pro Gln Asn Ile Leu Val Thr Ser Ser Gly Gln Ile Lys 145 150 155 160 Leu Ala Asp Phe Gly Leu Ala Arg Ile Tyr Ser Phe Gln Met Ala Leu 165 170 175 Thr Ser Val Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu 180 185 190 Gln Ser Ser Tyr Ala Thr Pro Val Asp Leu Trp Ser Val Gly Cys Ile 195 200 205 Phe Ala Glu Met Phe Arg Arg Lys Pro Leu Phe Arg Gly Ser Ser Asp 210 215 220 Val Asp Gln Leu Gly Lys Ile Leu Asp Ile Ile Gly Leu Pro Gly Glu 225 230 235 240 Glu Asp Trp Pro Arg Asp Val Ala Leu Pro Arg Gln Ala Phe His Ser 245 250 255 Lys Ser Ala Gln Pro Ile Glu Lys Phe Val Thr Asp Ile Asp Glu Leu 260 265 270 Gly Lys Asp Leu Leu Leu Lys Cys Leu Thr Phe Asn Pro Ala Lys Arg 275 280 285 Ile Ser Ala Tyr Gly Ala Leu Asn His Pro Tyr Phe Gln Asp Leu Glu 290 295 300 Arg Tyr Lys Asp Asn Leu Asn Ser His Leu Pro Ser Asn Gln Ser Thr 305 310 315 320 Ser Glu Leu Asn Thr Ala 325 22 2470 DNA Mus musculus (Balb/C) 22 ggcagcgagt gagcaccccg gttccactgt gccgcacccg cagcctgaag ccagcatgga 60 gaaggacagc ctgagtcgcg ccgatcagca gtatgagtgc gtggcggaga tcggcgaagg 120 cgcctatggg aaggtgttca aggcccgcga cctgaagaac ggcggccgct tcgtggctct 180 gaagcgcgtg cgagtgcaga ccagtgagga gggcatgccg ctctccacca tccgcgaggt 240 ggcggtgctg aggcacctgg agaccttcga gcaccccaac gtggtcaggt tgtttgatgt 300 gtgcacagtg tcacggacgg acagagaaac caagcttaca ctagtgtttg agcatgttga 360 tcaagacttg accacttact tggataaagt tccagagccc ggcgtaccca cagaaaccat 420 aaaggatatg atgtttcagc ttctccgagg tctggacttt cttcattctc acagagtagt 480 gcatcgtgat ctgaaaccgc agaacattct ggtgaccagc agtggacaga taaagctggc 540 tgactttggc cttgcccgca tctatagttt tcagatggcc cttacctcgg tggtcgtcac 600 gctgtggtac cgagccccag aagtcctgct ccagtccagc tatgccaccc ctgtggacct 660 ctggagtgtc ggttgcatct ttgcagaaat gtttcgcaga aagcctcttt ttcgtggaag 720 ttcagacgtg gatcaactag gaaaaatctt ggacatcatt ggactcccag gagaggaaga 780 ctggcctagg gacgtggccc ttccccggca ggcttttcat tccaaatctg ctcaacccat 840 cgagaagttt gtgacagata ttgacgaact aggcaaagac ctacttctga aatgcctgac 900 gtttaatcca gctaaaagga tatccgccta cggcgccctg aatcacccgt acttccaaga 960 tctggagaga tacaaggaca acctgaactc tcacctgcca tccaaccaga gcacctcgga 1020 gctgaacaca gcctgaggtt ccacggggat gcccatgagc tcgtcatctg aacacattgg 1080 cggctgcgag tcccctaagc aagcctctca gagcagttga agattgctgg ctgccaacct 1140 tctggctgcc agcttctggg tgggctctgc cttaccaagg aaaccaccta gtttactgtt 1200 cagagatcaa tgcaagggtg attgcagctt tatgttcgtt tgtacacttg tttgttttgt 1260 ctgtttgttt caagaacctg gaaaacttcc agaagaagag aagctgctga ccaattgtgc 1320 tgccatttcg ttttctaacc ttgaatgctg ccagtgtagg gtgggaatcc aggcccagct 1380 gagttatgat gtaatccgcc tgcagctgct gggcctgctt tggtacttgt gagtgtgtgt 1440 gcatgcgtat gtgtgtgtaa gagagaagag gaggggagag aaagacccct gatctcgtca 1500 agtgttactt tttttttgta gaaaacaaga ataattgagt tttaaagagt agaggtgact 1560 gatagtaaga agggcttgct cagtgaaagg tgattcacaa tggagtcttg ttaggaaggt 1620 tggacctaag tcctcagagt tgccttcctg tccaaaagct tttgctagca gtaaacaata 1680 aaggtttaga tgccacaaaa aatgggggga accgcaatat tttttaagag actttttaag 1740 gcatacatct tctatttact ctttggaaag ctgaacttaa tgtgtcccag gccctatata 1800 tagtacagta tgtacttaat tgtttctttg gggaaagatg ctataagtat cttattactt 1860 gcaatacatt taaggagtga gtgtacctca gataggtttt aaagatagag agcacctgtt 1920 ttctggtgtg agatgttatc attttcttca cgtctcttga taccttgata ccttgtcacc 1980 ttagggaatc acttcctgct ctgactagag gcgggaatac catctagctg tctccaccac 2040 ccaccatggc gcatctgcct tgtgctgcct tgtgtagtgc gaagctctca accaccagca 2100 cttctaattc attttcctgc cactgcctgg ctaacgacag atggcccagc tgccccaatc 2160 ccacacccgc ttgcacgctt accgtctttc accgaatgct ttgggcgtag gctcccattc 2220 cgaaacccta acagtatccc cttgtgcctt tgtaatacag tcttccccct gccgcagctg 2280 aggtcaccta ggcagtgaag agtgcttgtt ctgtgtgtgt atagactact accgactgtc 2340 acttggtgtt tcctatcttt aagtgtatgt tgtcagtgta atgtctgagg aaatgtcttt 2400 tcctctcttc tagagataac tacttactct ctaaagtgat ctctctgtct gtccgcagga 2460 tgtgtttctg 2470 23 111 PRT Rattus Norvegicus 23 Gly Lys Val Phe Lys Ala Arg Asp Leu Lys Asn Gly Gly Arg Phe Val 1 5 10 15 Ala Leu Lys Arg Val Arg Val Gln Thr Gly Glu Glu Gly Met Pro Leu 20 25 30 Ser Thr Ile Arg Glu Val Ala Val Leu Arg His Leu Glu Thr Phe Glu 35 40 45 His Pro Asn Val Val Arg Leu Phe Asp Val Cys Thr Val Ser Arg Thr 50 55 60 Asp Arg Glu Thr Lys Leu Thr Leu Val Phe Glu His Val Asp Gln Asp 65 70 75 80 Leu Thr Thr Tyr Leu Asp Lys Val Pro Glu Pro Gly Val Pro Thr Glu 85 90 95 Thr Ile Lys Asp Met Met Phe Gln Leu Leu Arg Gly Leu Asp Phe 100 105 110 24 333 DNA Rattus Norvegicus 24 gggaaggtgt tcaaggcccg cgacctgaag aacggcggcc gcttcgtggc tctgaagcgc 60 gtgcgagtgc agaccggaga ggagggcatg ccgctctcca ccatccgcga ggtggcggtg 120 ctgaggcacc tggagacctt tgagcacccc aacgtggtca ggttgtttga cgtgtgcaca 180 gtgtcacgga cagacagaga aactaaactt acgctagtgt ttgagcatgt tgatcaagac 240 ttgaccactt acttggataa agttccagaa cccggtgtgc ccacagagac cataaaggat 300 atgatgtttc agcttctccg aggtctggac ttt 333 25 326 PRT Gallus gallus 25 Met Asp Lys Asp Gly Thr Asn Leu Ala Asp Gln Gln Tyr Glu Cys Val 1 5 10 15 Ala Glu Ile Gly Glu Gly Ala Tyr Gly Lys Val Phe Lys Ala Arg Asp 20 25 30 Leu Lys Asn Gly Gly Arg Phe Val Ala Leu Lys Arg Val Arg Val Gln 35 40 45 Thr Ser Glu Glu Gly Met Pro Leu Ser Thr Ile Arg Glu Val Ala Val 50 55 60 Leu Arg His Leu Glu Thr Phe Glu His Pro Asn Val Val Arg Leu Phe 65 70 75 80 Asp Val Cys Thr Val Ser Arg Thr Asp Arg Glu Thr Lys Leu Thr Leu 85 90 95 Val Phe Glu His Val Asp Gln Asp Leu Thr Thr Tyr Leu Asp Lys Val 100 105 110 Pro Glu Pro Gly Val Pro Thr Glu Thr Ile Lys Asp Met Met Leu Gln 115 120 125 Leu Phe Arg Gly Leu Asp Phe Leu His Ser His Arg Val Val His Arg 130 135 140 Asp Leu Lys Pro Gln Asn Ile Leu Val Thr Ser Ser Gly Gln Ile Lys 145 150 155 160 Leu Ala Asp Phe Gly Leu Ala Arg Ile Tyr Ser Phe Gln Met Ala Leu 165 170 175 Thr Ser Val Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu 180 185 190 Gln Ser Ser Tyr Ala Thr Pro Val Asp Leu Trp Ser Val Gly Cys Ile 195 200 205 Phe Ala Glu Met Phe Arg Arg Lys Pro Leu Phe Arg Gly Asn Ser Asp 210 215 220 Val Asp Gln Leu Gly Lys Ile Phe Asp Val Ile Gly Leu Pro Glu Glu 225 230 235 240 Glu Asp Trp Pro Asn Asp Val Ala Leu Pro Arg Asn Ala Phe Ala Ser 245 250 255 Arg Pro Ala Gln Pro Ile Glu Lys Phe Val Pro Asp Ile Asp Asp Met 260 265 270 Gly Lys Asp Leu Leu Leu Lys Cys Leu Ala Phe Asn Pro Ala Lys Arg 275 280 285 Ile Ser Ala Tyr Ala Ala Leu Ser His Pro Tyr Phe His Asp Leu Glu 290 295 300 Lys Cys Lys Glu Asn Leu Asp Ser His Met Ser Ser Ser Gln Asn Ser 305 310 315 320 Ser Glu Val Asn Ala Ser 325 26 1453 DNA Gallus gallus 26 gccgccggcg gagcgggcgg ccgcggcgcg caccagatct agctgccttc gcacagacca 60 tgctcccaaa ggctgtatga tcatttctgg tgactgccag cggaggctca ctgggacccg 120 tgcagcagag aaggcggacg aggggttcct gctgcccctc gggcagaggt accggcgtgt 180 ggccgaccca cagcgaggtg ccagcttcag aagccggcag agacgagagc tgccgtgagg 240 tgctccggac ccgggaggaa gcggagcggc ggcagcatgg acaaggacgg caccaacctg 300 gccgaccaac agtatgagtg cgtggctgag atcggcgaag gagcctacgg gaaggtgttc 360 aaggcccgcg acctgaagaa tggcggccgc ttcgtggcgc tgaagcgggt gcgggtgcag 420 accagcgagg agggcatgcc gctgtccacc atccgggagg tggccgtcct gaggcacctg 480 gagaccttcg agcaccccaa cgtggtcaga ttgtttgatg tgtgcaccgt gtcacgaaca 540 gacagagaaa ccaagttaac gttggtgttt gaacatgtgg atcaagactt gactacttac 600 ttggataaag ttccagagcc tggagtgcct actgaaacta taaaggatat gatgcttcag 660 ctgtttcggg gactggattt tctgcattca catcgtgtgg tgcatcggga cctgaaaccc 720 cagaatatcc ttgtaaccag cagtgggcag ataaagttag ctgactttgg acttgcacga 780 atctacagtt ttcagatggc tcttacatca gtggttgtta ctttgtggta tagagctcct 840 gaagttttgc ttcagtccag ctatgcaaca ccagttgatc tttggagtgt tggttgcata 900 tttgcagaaa tgttccgtcg aaaaccactc ttccgtggaa attcagatgt tgatcagcta 960 ggaaaaatct ttgatgtaat tggactccca gaagaagagg actggcctaa tgatgtggcc 1020 cttccaagaa atgcttttgc ttccagaccc gcacaaccta ttgaaaaatt tgtaccagat 1080 attgatgaca tgggcaaaga cttgcttctt aaatgcttag cgttcaatcc agccaagaga 1140 atatctgcct atgctgccct gtctcacccc tatttccatg atctggagaa atgcaaggag 1200 aatctggact ctcacatgtc atccagccaa aactccagtg aggtgaacgc atcataatgc 1260 tgactgaaga cgaacctatg aatgaacaag atgtgtagct gacaaggcca ctgtcccata 1320 caaaccacat ggctgttcta gtgaagatca ttgtttggag gttttacaca cctgtctttc 1380 attttgggat tgcgatgtgc ttatccaagg aaatcaacgt agtttacttt catcagagca 1440 atgcaagggc cag 1453 27 298 PRT Homo sapiens VARIANT (1)...(298) Xaa = Any Amino Acid 27 Met Glu Asn Phe Gln Lys Val Glu Lys Ile Gly Glu Gly Thr Tyr Gly 1 5 10 15 Val Val Tyr Lys Ala Arg Asn Lys Leu Thr Gly Glu Val Val Ala Leu 20 25 30 Lys Lys Ile Arg Xaa Asp Thr Glu Thr Glu Gly Val Pro Ser Thr Ala 35 40 45 Ile Arg Glu Ile Ser Leu Leu Lys Glu Leu Asn His Pro Asn Ile Val 50 55 60 Lys Leu Leu Asp Val Ile His Thr Glu Asn Lys Leu Tyr Leu Val Phe 65 70 75 80 Glu Phe Leu His Gln Asp Leu Lys Lys Phe Met Asp Ala Ser Ala Leu 85 90 95 Thr Gly Ile Pro Leu Pro Leu Ile Lys Ser Tyr Leu Phe Gln Leu Leu 100 105 110 Gln Gly Leu Ala Phe Cys His Ser His Arg Val Leu His Arg Asp Leu 115 120 125 Lys Pro Gln Asn Leu Leu Ile Asn Thr Glu Gly Ala Ile Lys Leu Ala 130 135 140 Asp Phe Gly Leu Ala Arg Ala Phe Gly Val Pro Val Arg Thr Tyr Thr 145 150 155 160 His Glu Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Ile Leu Leu Gly 165 170 175 Cys Lys Tyr Tyr Ser Thr Ala Val Asp Ile Trp Ser Leu Gly Cys Ile 180 185 190 Phe Ala Glu Met Val Thr Arg Arg Ala Leu Phe Pro Gly Asp Ser Glu 195 200 205 Ile Asp Gln Leu Phe Arg Ile Phe Arg Thr Leu Gly Thr Pro Asp Glu 210 215 220 Val Val Trp Pro Gly Val Thr Ser Met Pro Asp Tyr Lys Pro Ser Phe 225 230 235 240 Pro Lys Trp Ala Arg Gln Asp Phe Ser Lys Val Val Pro Pro Leu Asp 245 250 255 Glu Asp Gly Arg Ser Leu Leu Ser Gln Met Leu His Tyr Asp Pro Asn 260 265 270 Lys Arg Ile Ser Ala Lys Ala Ala Leu Ala His Pro Phe Phe Gln Asp 275 280 285 Val Thr Lys Pro Val Pro His Leu Arg Leu 290 295 28 1297 DNA Homo sapiens misc_feature (1)...(1297) n = A,T,C or G 28 cgttggccaa attgacaaga gcgagaggta tactgcgttc catcccgacc nggggccacg 60 gtactgggcc ctgtttcccc ctcctcggcc cccgagagcc agggtccgcc ttctgcaggg 120 ttcccaggcc cccgctccag ggccgggctg acccgactcg ctggcgcttc atggagaact 180 tccaaaaggt ggaaaagatc ggagagggca cgtacggagt tgtgtacaaa gccagaaaca 240 agttgacggg agaggtggtg gcgcttaaga aaatccgnnt ggacactgag actgagggtg 300 tgcccagtac tgccatccga gagatctctc tgcttaagga gcttaaccat cctaatattg 360 tcaagctgct ggatgtcatt cacacagaaa ataaactcta cctggttttt gaatttctgc 420 accaagatct caagaaattc atggatgcct ctgctctcac tggcattcct cttcccctca 480 tcaagagcta tctgttccag ctgctccagg gcctagcttt ctgccattct catcgggtcc 540 tccaccgaga ccttaaacct cagaatctgc ttattaacac agagggggcc atcaagctag 600 cagactttgg actagccaga gcttttggag tccctgttcg tacttacacc catgaggtgg 660 tgaccctgtg gtaccgagct cctgaaatcc tcctgggctg caaatattat tccacagctg 720 tggacatctg gagcctgggc tgcatctttg ctgagatggt gactcgccgg gccctattcc 780 ctggagattc tgagattgac cagctcttcc ggatctttcg gactctgggg accccagatg 840 aggtggtgtg gccaggagtt acttctatgc ctgattacaa gccaagtttc cccaagtggg 900 cccggcaaga ttttagtaaa gttgtacctc ccctggatga agatggacgg agcttgttat 960 cgcaaatgct gcactacgac cctaacaagc ggatttcggc caaggcagcc ctggctcacc 1020 ctttcttcca ggatgtgacc aagccagtac cccatcttcg actctgatag ccttcttgaa 1080 gcccccagcc ctaatctcac cctctcctcc agtgtgggct tgaccaggct tggccttggg 1140 ctatttggac tcaggtgggc cctctgaact tgccttaaac actcaccttc tagtcttggc 1200 cagccaactc tgggaataca ggggtgaaag gggggaacca gtgaaaatga aaggaagttt 1260 cagtattaga ttgcacttaa gttagcctcc accaccc 1297 29 298 PRT Homo sapiens VARIANT (1)...(298) Xaa = Any Amino Acid 29 Met Glu Asn Phe Gln Lys Val Glu Lys Ile Gly Glu Gly Thr Tyr Gly 1 5 10 15 Val Val Tyr Lys Ala Cys Asn Lys Leu Thr Gly Glu Val Val Ala Leu 20 25 30 Lys Lys Ile Arg Xaa Asp Thr Glu Thr Glu Gly Val Pro Ser Thr Ala 35 40 45 Ile Arg Glu Ile Ser Leu Leu Lys Glu Leu Asn His Pro Asn Ile Val 50 55 60 Lys Leu Leu Asp Val Ile His Thr Glu Asn Lys Leu Tyr Leu Val Phe 65 70 75 80 Glu Phe Leu His Gln Asp Leu Lys Lys Phe Met Asp Ala Ser Ala Leu 85 90 95 Thr Gly Ile Pro Leu Pro Leu Ile Lys Ser Tyr Leu Phe Gln Leu Leu 100 105 110 Gln Gly Leu Ala Phe Cys His Ser His Arg Val Leu His Arg Asp Leu 115 120 125 Lys Pro Gln Asn Leu Leu Ile Asn Thr Glu Gly Ala Ile Lys Leu Ala 130 135 140 Asp Phe Gly Leu Ala Arg Ala Phe Gly Val Pro Val Arg Thr Tyr Thr 145 150 155 160 His Glu Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Ile Leu Leu Gly 165 170 175 Cys Lys Tyr Tyr Ser Thr Ala Val Asp Ile Trp Ser Leu Gly Cys Ile 180 185 190 Phe Ala Glu Met Val Thr Arg Arg Ala Leu Phe Pro Gly Asp Ser Glu 195 200 205 Ile Asp Gln Leu Phe Arg Ile Phe Arg Thr Leu Gly Thr Pro Asp Glu 210 215 220 Val Val Trp Pro Gly Val Thr Ser Met Pro Asp Tyr Lys Pro Ser Phe 225 230 235 240 Pro Lys Trp Ala Arg Gln Asp Phe Ser Lys Val Val Pro Pro Leu Asp 245 250 255 Glu Asp Gly Arg Ser Leu Leu Ser Gln Met Leu His Tyr Asp Pro Asn 260 265 270 Lys Arg Ile Ser Ala Lys Ala Ala Leu Ala His Pro Phe Phe Gln Asp 275 280 285 Val Thr Lys Pro Val Pro His Leu Arg Leu 290 295 30 1297 DNA Artificial Sequence Mutation 30 cgttggccaa attgacaaga gcgagaggta tactgcgttc catcccgacc nggggccacg 60 gtactgggcc ctgtttcccc ctcctcggcc cccgagagcc agggtccgcc ttctgcaggg 120 ttcccaggcc cccgctccag ggccgggctg acccgactcg ctggcgcttc atggagaact 180 tccaaaaggt ggaaaagatc ggagagggca cgtacggagt tgtgtacaaa gcctgtaaca 240 agttgacggg agaggtggtg gcgcttaaga aaatccgnnt ggacactgag actgagggtg 300 tgcccagtac tgccatccga gagatctctc tgcttaagga gcttaaccat cctaatattg 360 tcaagctgct ggatgtcatt cacacagaaa ataaactcta cctggttttt gaatttctgc 420 accaagatct caagaaattc atggatgcct ctgctctcac tggcattcct cttcccctca 480 tcaagagcta tctgttccag ctgctccagg gcctagcttt ctgccattct catcgggtcc 540 tccaccgaga ccttaaacct cagaatctgc ttattaacac agagggggcc atcaagctag 600 cagactttgg actagccaga gcttttggag tccctgttcg tacttacacc catgaggtgg 660 tgaccctgtg gtaccgagct cctgaaatcc tcctgggctg caaatattat tccacagctg 720 tggacatctg gagcctgggc tgcatctttg ctgagatggt gactcgccgg gccctattcc 780 ctggagattc tgagattgac cagctcttcc ggatctttcg gactctgggg accccagatg 840 aggtggtgtg gccaggagtt acttctatgc ctgattacaa gccaagtttc cccaagtggg 900 cccggcaaga ttttagtaaa gttgtacctc ccctggatga agatggacgg agcttgttat 960 cgcaaatgct gcactacgac cctaacaagc ggatttcggc caaggcagcc ctggctcacc 1020 ctttcttcca ggatgtgacc aagccagtac cccatcttcg actctgatag ccttcttgaa 1080 gcccccagcc ctaatctcac cctctcctcc agtgtgggct tgaccaggct tggccttggg 1140 ctatttggac tcaggtgggc cctctgaact tgccttaaac actcaccttc tagtcttggc 1200 cagccaactc tgggaataca ggggtgaaag gggggaacca gtgaaaatga aaggaagttt 1260 cagtattaga ttgcacttaa gttagcctcc accaccc 1297 31 298 PRT Mus musculus 31 Met Glu Asn Phe Gln Lys Val Glu Lys Ile Gly Glu Gly Thr Tyr Gly 1 5 10 15 Val Val Tyr Lys Ala Lys Asn Lys Leu Thr Gly Glu Val Val Ala Leu 20 25 30 Lys Lys Ile Arg Leu Asp Thr Glu Thr Glu Gly Val Pro Ser Thr Ala 35 40 45 Ile Arg Glu Ile Ser Leu Leu Lys Glu Leu Asn His Pro Asn Ile Val 50 55 60 Lys Leu Leu Asp Val Ile His Thr Glu Asn Lys Leu Tyr Leu Val Phe 65 70 75 80 Glu Phe Leu His Gln Asp Leu Lys Lys Phe Met Asp Ala Ser Ala Leu 85 90 95 Thr Gly Ile Pro Leu Pro Leu Ile Lys Ser Tyr Leu Phe Gln Leu Leu 100 105 110 Gln Gly Leu Ala Phe Cys His Ser His Arg Val Leu His Arg Asp Leu 115 120 125 Lys Pro Gln Asn Leu Leu Ile Asn Ala Glu Gly Ser Ile Lys Leu Ala 130 135 140 Asp Phe Gly Leu Ala Arg Ala Phe Gly Val Pro Val Arg Thr Tyr Thr 145 150 155 160 His Glu Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Ile Leu Leu Gly 165 170 175 Cys Lys Tyr Tyr Ser Thr Ala Val Asp Ile Trp Ser Leu Gly Cys Ile 180 185 190 Phe Ala Glu Met Val Thr Arg Arg Ala Leu Phe Pro Gly Asp Ser Glu 195 200 205 Ile Asp Gln Leu Phe Arg Ile Phe Arg Thr Leu Gly Thr Pro Asp Glu 210 215 220 Val Val Trp Pro Gly Val Thr Ser Met Pro Asp Tyr Lys Pro Ser Phe 225 230 235 240 Pro Lys Trp Ala Arg Gln Asp Phe Ser Lys Val Val Pro Pro Leu Asp 245 250 255 Glu Asp Gly Arg Ser Leu Leu Ser Gln Met Leu His Tyr Asp Pro Asn 260 265 270 Lys Arg Ile Ser Ala Lys Ala Ala Leu Ala His Pro Phe Phe Gln Asp 275 280 285 Val Thr Lys Pro Val Pro His Leu Arg Leu 290 295 32 1708 DNA Mus musculus 32 caagggctga gctctccttg cgttccatcc cgagctggga ctgccgtgct cacccggggt 60 cccccaggtc ctccgctccg agtgtcgggc ccgctttcgt cagggttccc gggccccgct 120 cccgggggcc tgagccgcct cactagcgct ccatggagaa ctttcaaaag gtggagaaga 180 ttggagaggg cacgtacgga gtggtgtaca aagccaaaaa caagttgacg ggagaagttg 240 tggcgcttaa gaagatccgg ctcgacactg agactgaagg tgtacccagt actgccatcc 300 gagagatctc tctccttaag gaacttaatc accctaatat cgtcaagctg ctggatgtca 360 tccacacaga aaataagctt tatctggttt ttgaatttct gcaccaggac ctcaagaaat 420 tcatggatgc ctctgctctc acgggcattc ctcttcccct catcaagagc tatctgttcc 480 agctgctcca gggcctggct ttctgccatt ctcaccgtgt ccttcaccga gaccttaagc 540 cccagaacct gcttatcaat gcagaggggt ccatcaagct ggcagacttt ggactagcaa 600 gagcctttgg agtccctgtc cgaacttaca ctcatgaggt ggtgaccctg tggtaccgag 660 cacctgaaat tcttctgggc tgcaagtact actccacagc cgtggatatc tggagcctgg 720 gctgcatctt tgctgaaatg gtgacccgca gggccctatt ccctggagat tctgagattg 780 accaactctt ccggatcttt cggactctgg ggaccccaga tgaggtggtt tggccaggag 840 ttacttctat gcctgattat aagccaagtt tccccaagtg ggctcggcaa gattttagca 900 aagttgtgcc tcccctggat gaagatggac ggagcttgtt atcgcaaatg ctgcactatg 960 accccaacaa gcggatttca gccaaagcag ccctggctca ccctttcttc caggatgtaa 1020 ctaaaccagt gccccacctt cggctctgat gcccttccca aagccctttt cacccgtggt 1080 ctgacttgac cctgggcctt ttggacacag gcaagaagag ccaggagggc acagggcttg 1140 cacgtcactc tggtctgttc atcgtggttc acagggcaag gtgaaagaca cttgaatttc 1200 tctttttagc aatcttactg ttttctggtg ctgactgcta cacccaggac tttgccctca 1260 ctaagcaaat gtaccaccaa tgagccaggt tcctagcctt ccttttggaa gctcagttct 1320 tgagttgtca gagggcctca ttgccgtcct cctctgagag cagtgatgca ggccaggaga 1380 catcatttga gaatgctgat gcttttgtaa ggctttgacc tggttaggtc attgggggaa 1440 attttctata aaagagtgaa caattatatt tatatttcaa gttaaagtag tttggatact 1500 ttagtggttt tgttgttgtt tcttcttttt cctttttttt ttttttttgt cagtgtggat 1560 ggatttgttg ccatgtgcac tttgggattt tgtaattgtt aaaagaaaat atttctttta 1620 tgattttctt ctcccactcc ctgtcctcca gtgttctgtt aatattattt gtaatttagt 1680 ttgtaaattc attaaaagaa aatattct 1708 33 346 PRT Mus musculus 33 Met Glu Asn Phe Gln Lys Val Glu Lys Ile Gly Glu Gly Thr Tyr Gly 1 5 10 15 Val Val Tyr Lys Ala Lys Asn Lys Leu Thr Gly Glu Val Val Ala Leu 20 25 30 Lys Lys Ile Arg Leu Asp Thr Glu Thr Glu Gly Val Pro Ser Thr Ala 35 40 45 Ile Arg Glu Ile Ser Leu Leu Lys Glu Leu Asn His Pro Asn Ile Val 50 55 60 Lys Leu Leu Asp Val Ile His Thr Glu Asn Lys Leu Tyr Leu Val Phe 65 70 75 80 Glu Phe Leu His Gln Asp Leu Lys Lys Phe Met Asp Ala Ser Ala Leu 85 90 95 Thr Gly Ile Pro Leu Pro Leu Ile Lys Ser Tyr Leu Phe Gln Leu Leu 100 105 110 Gln Gly Leu Ala Phe Cys His Ser His Arg Val Leu His Arg Asp Leu 115 120 125 Lys Pro Gln Asn Leu Leu Ile Asn Ala Glu Gly Ser Ile Lys Leu Ala 130 135 140 Asp Phe Gly Leu Ala Arg Ala Phe Gly Val Pro Val Arg Thr Tyr Thr 145 150 155 160 His Glu Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Ile Leu Leu Gly 165 170 175 Cys Lys Tyr Tyr Ser Thr Ala Val Asp Ile Trp Ser Leu Gly Cys Ile 180 185 190 Phe Ala Glu Met His Leu Val Cys Thr Gln His His Ala Lys Cys Cys 195 200 205 Gly Glu His Arg Arg Asn Gly Arg His Ser Leu Cys Pro Leu Cys Ser 210 215 220 Tyr Leu Glu Val Ala Ala Ser Gln Gly Gly Gly Met Thr Ala Val Ser 225 230 235 240 Ala Pro His Pro Val Thr Arg Arg Ala Leu Phe Pro Gly Asp Ser Glu 245 250 255 Ile Asp Gln Leu Phe Arg Ile Phe Arg Thr Leu Gly Thr Pro Asp Glu 260 265 270 Val Val Trp Pro Gly Val Thr Ser Met Pro Asp Tyr Lys Pro Ser Phe 275 280 285 Pro Lys Trp Ala Arg Gln Asp Phe Ser Lys Val Val Pro Pro Leu Asp 290 295 300 Glu Asp Gly Arg Ser Leu Leu Ser Gln Met Leu His Tyr Asp Pro Asn 305 310 315 320 Lys Arg Ile Ser Ala Lys Ala Ala Leu Ala His Pro Phe Phe Gln Asp 325 330 335 Val Thr Lys Pro Val Pro His Leu Arg Leu 340 345 34 1380 DNA Mus musculus 34 tcccgagctg gggactgccg ggctcacccg gggtccccca ggtcctccgc tccgagggtc 60 gggcccgctt tcgtcagggt tcccgggccc cgctcccggg ggcctgagcc gcctcactag 120 cgctccatgg agaacttcca aaaggtggag aagattggag agggcacgta cggagtggtg 180 tacaaagcca aaaacaagtt gacgggagaa gttgtggcgc ttaagaagat ccggctcgac 240 actgagactg aaggtgtacc cagtactgcc atccgagaga tctctctcct taaggaactt 300 aatcacccta atatcgtcaa gctgctggat gtcatccaca cagaaaataa gctttatctg 360 gtttttgaat ttctgcacca ggacctcaag aaattcatgg atgcctctgc tctcacgggc 420 attcctcttc ccctcatcaa gagctatctg ttccagctgc tccagggcct ggctttctgc 480 cattctcacc gtgtccttca ccgagacctt aagccccaga acctgcttat caatgcagag 540 gggtccatca agctggcaga ctttggacta gcaagagcct ttggagtccc tgtccgaact 600 tacactcatg aggtggtgac cctgtggtac cgagcacctg aaattcttct gggctgcaag 660 tactactcca cagccgtgga tatctggagc ctgggctgca tctttgctga aatgcaccta 720 gtgtgtaccc agcaccatgc taagtgctgt ggggaacaca gaagaaatgg aagacacagt 780 ctctgcccgc tgtgctccta tctagaagtg gctgcatcac aaggaggggg gatgaccgca 840 gtgtctgccc cacaccccgt gacccgcagg gccctattcc ctggagattc tgagattgac 900 caactcttcc ggatctttcg gactctgggg accccagatg aggtggtttg gccaggagtt 960 acttctatgc ctgattataa gccaagtttc cccaagtggg ctcggcaaga ttttagcaaa 1020 gttgtgcctc ccctggatga agatggacgg agcttgttat cgcaaatgct gcactatgac 1080 cccaacaagc ggatttcagc caaagcagcc ctggctcacc ctttcttcca ggatgtaact 1140 aaaccagtgc cccaccttcg gctctgatgc ccttcccaaa gcccttttca cccgtggtct 1200 gacttgaccc tgggcctttt ggacacaggt cagccttctg atgttttctg gctgtcttag 1260 catccgcctt ttctcttgcc agccagttct ggggattcag aggtgcaggg aagggttagg 1320 gaaaaggggg caaggttttg ggaattagat gcacttaacc cggttccacc agttctccct 1380 35 298 PRT Mesocricetus auratus 35 Met Glu Asn Phe Gln Lys Val Glu Lys Ile Gly Glu Gly Thr Tyr Gly 1 5 10 15 Val Val Tyr Lys Ala Lys Asn Lys Leu Thr Gly Glu Val Val Ala Leu 20 25 30 Lys Lys Ile Arg Leu Asp Thr Glu Thr Glu Gly Val Pro Ser Thr Ala 35 40 45 Ile Arg Glu Ile Ser Leu Leu Lys Glu Leu Asn His Pro Asn Ile Val 50 55 60 Lys Leu Leu Asp Val Ile His Thr Glu Asn Lys Leu Tyr Leu Val Phe 65 70 75 80 Glu Leu Leu His Gln Asp Leu Lys Lys Phe Met Asp Ala Ser Ala Val 85 90 95 Thr Gly Ile Pro Leu Pro Leu Ile Lys Ser Tyr Leu Phe Gln Leu Leu 100 105 110 Gln Gly Leu Ala Phe Cys His Ser His Arg Val Leu His Arg Asp Leu 115 120 125 Lys Pro Gln Asn Leu Leu Ile Asn Ala Glu Gly Ser Ile Lys Leu Ala 130 135 140 Asp Phe Gly Leu Ala Arg Ala Phe Gly Val Pro Val Arg Thr Tyr Thr 145 150 155 160 His Glu Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Ile Leu Leu Gly 165 170 175 Cys Lys Tyr Tyr Ser Thr Ala Val Asp Ile Trp Ser Leu Gly Cys Ile 180 185 190 Phe Ala Glu Met Val Thr Arg Arg Ala Leu Phe Pro Gly Asp Ser Glu 195 200 205 Ile Asp Gln Leu Phe Arg Ile Phe Arg Thr Leu Gly Thr Pro Asp Glu 210 215 220 Val Val Trp Pro Gly Val Thr Ser Met Pro Asp Tyr Lys Pro Ser Phe 225 230 235 240 Pro Lys Trp Ala Arg Gln Asp Phe Ser Lys Val Val Pro Pro Leu Asp 245 250 255 Glu Asp Gly Arg Ser Leu Leu Ser Gln Met Leu His Tyr Asp Pro Asn 260 265 270 Lys Arg Ile Ser Ala Lys Ala Ala Leu Ala His Pro Phe Phe Gln Asp 275 280 285 Val Thr Lys Pro Val Pro His Leu Arg Leu 290 295 36 1065 DNA Mesocricetus auratus 36 catcccgagc tggggctgcc gtgctcacct tggcccccag gtcctccgcc cctgagtgtc 60 gggcccgctt tctttagggt tcccgggccc cgctcccggg ggcctgagcc gccgcattag 120 cgctccatgg agaacttcca aaaggtggag aagatcggag agggcacgta cggagtggtg 180 tacaaagcca aaaacaagtt gacgggagaa gtggtggcgc ttaagaaaat ccggctcgac 240 actgagactg aaggtgtgcc cagtactgcc atccgagaga tctctctgct taaggaactt 300 aatcacccta atattgtcaa gctgctggat gtcatccaca cagaaaataa gctttacctg 360 gttttcgagc ttctgcacca ggacctcaag aaatttatgg atgcctctgc tgtcactggc 420 atccctcttc ccctcatcaa gagctatctg ttccagctgc tccagggcct agcattctgc 480 cattctcacc gggtcctgca ccgagacctt aagccccaga atctgcttat caatgcagag 540 ggatccatca agttggcaga ctttggacta gcaagagcct ttggagtccc cgttcgtact 600 tatactcatg aggtggtgac cttgtggtac cgagcacctg aaatccttct aggctgcaag 660 tactactcca cagccgtgga tatctggagc ctgggctgca tcttcgctga aatggtgacc 720 cggcgggccc tgtttcctgg agattctgag attgatcaac tcttccggat ctttcggact 780 ctggggaccc cagatgaggt ggtttggcca ggagttactt ctatgcctga ttataagccg 840 agtttcccca agtgggctcg gcaagatttt agcaaagttg tgcctccact ggatgaagat 900 ggacggagcc tgttatcgca aatgctgcac tatgacccca acaagcggat ttcagccaaa 960 gcagccctgg cccacccttt cttccaggat gtgactaagc cagtacccca ccttcggctg 1020 tgatgtcctt cccaaagccc tcctcaccct tgggtcttga cttga 1065 37 346 PRT Mesocricetus auratus 37 Met Glu Asn Phe Gln Lys Val Glu Lys Ile Gly Glu Gly Thr Tyr Gly 1 5 10 15 Val Val Tyr Lys Ala Lys Asn Lys Leu Thr Gly Glu Val Val Ala Leu 20 25 30 Lys Lys Ile Arg Leu Asp Thr Glu Thr Glu Gly Val Pro Ser Thr Ala 35 40 45 Ile Arg Glu Ile Ser Leu Leu Lys Glu Leu Asn His Pro Asn Ile Val 50 55 60 Lys Leu Leu Asp Val Ile His Thr Glu Asn Lys Leu Tyr Leu Val Phe 65 70 75 80 Glu Leu Leu His Gln Asp Leu Lys Lys Phe Met Asp Ala Ser Ala Val 85 90 95 Thr Gly Ile Pro Leu Pro Leu Ile Lys Ser Tyr Leu Phe Gln Leu Leu 100 105 110 Gln Gly Leu Ala Phe Cys His Ser His Arg Val Leu His Arg Asp Leu 115 120 125 Lys Pro Gln Asn Leu Leu Ile Asn Ala Glu Gly Ser Ile Lys Leu Ala 130 135 140 Asp Phe Gly Leu Ala Arg Ala Phe Gly Val Pro Val Arg Thr Tyr Thr 145 150 155 160 His Glu Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Ile Leu Leu Gly 165 170 175 Cys Lys Tyr Tyr Ser Thr Ala Val Asp Ile Trp Ser Leu Gly Cys Ile 180 185 190 Phe Ala Glu Met His Leu Val Cys Thr Gln His His Ala Lys Cys Cys 195 200 205 Gly Glu His Arg Arg Asn Gly Arg His Ser Leu Cys Pro Leu Cys Ser 210 215 220 Tyr Leu Glu Val Ala Ala Ser Gln Gly Gly Gly Met Thr Ala Val Ser 225 230 235 240 Thr Pro Tyr Pro Val Thr Arg Arg Ala Leu Phe Pro Gly Asp Ser Glu 245 250 255 Ile Asp Gln Leu Phe Arg Ile Phe Arg Thr Leu Gly Thr Pro Asp Glu 260 265 270 Val Val Trp Pro Gly Val Thr Ser Met Pro Asp Tyr Lys Pro Ser Phe 275 280 285 Pro Lys Trp Ala Arg Gln Asp Phe Ser Lys Val Val Pro Pro Leu Asp 290 295 300 Glu Asp Gly Arg Ser Leu Leu Ser Gln Met Leu His Tyr Asp Pro Asn 305 310 315 320 Lys Arg Ile Ser Ala Lys Ala Ala Leu Ala His Pro Phe Phe Gln Asp 325 330 335 Val Thr Lys Pro Val Pro His Leu Arg Leu 340 345 38 1209 DNA Mesocricetus auratus 38 catcccgagc tggggctgcc gtgctcacct tggcccccag gtcctccgcc cctgagtgtc 60 gggcccgctt tctttagggt tcccgggccc cgctcccggg ggcctgagcc gccgcattag 120 cgctccatgg agaacttcca aaaggtggag aagatcggag agggcacgta cggagtggtg 180 tacaaagcca aaaacaagtt gacgggagaa gtggtggcgc ttaagaaaat ccggctcgac 240 actgagactg aaggtgtgcc cagtactgcc atccgagaga tctctctgct taaggaactt 300 aatcacccta atattgtcaa gctgctggat gtcatccaca cagaaaataa gctttacctg 360 gttttcgagc ttctgcacca ggacctcaag aaatttatgg atgcctctgc tgtcactggc 420 atccctcttc ccctcatcaa gagctatctg ttccagctgc tccagggcct agcattctgc 480 cattctcacc gggtcctgca ccgagacctt aagccccaga atctgcttat caatgcagag 540 ggatccatca agttggcaga ctttggacta gcaagagcct ttggagtccc cgttcgtact 600 tatactcatg aggtggtgac cttgtggtac cgagcacctg aaatccttct aggctgcaag 660 tactactcca cagccgtgga tatctggagc ctgggctgca tcttcgctga aatgcaccta 720 gtgtgtaccc agcaccatgc taagtgctgt ggggaacaca gaagaaatgg aagacacagt 780 ctctgcccgc tgtgctccta tctagaagtg gctgcgtcac aaggaggggg gatgaccgca 840 gtgtctaccc cataccccgt gacccggcgg gccctgtttc ctggagattc tgagattgat 900 caactcttcc ggatctttcg gactctgggg accccagatg aggtggtttg gccaggagtt 960 acttctatgc ctgattataa gccgagtttc cccaagtggg ctcggcaaga ttttagcaaa 1020 gttgtgcctc cactggatga agatggacgg agcctgttat cgcaaatgct gcactatgac 1080 cccaacaagc ggatttcagc caaagcagcc ctggcccacc ctttcttcca ggatgtgact 1140 aagccagtac cccaccttcg gctgtgatgt ccttcccaaa gccctcctca cccttgggtc 1200 ttgacttga 1209 39 298 PRT Cricetulus griseus 39 Met Glu Asn Phe Gln Lys Val Glu Lys Ile Gly Glu Gly Thr Tyr Gly 1 5 10 15 Val Val Tyr Lys Ala Lys Asn Lys Leu Thr Gly Glu Val Val Ala Leu 20 25 30 Lys Lys Ile Arg Leu Asp Thr Glu Thr Glu Gly Val Pro Ser Thr Ala 35 40 45 Ile Arg Glu Ile Ser Leu Leu Lys Glu Leu Asn His Pro Asn Ile Val 50 55 60 Lys Leu Leu Asp Val Ile His Thr Glu Asn Lys Leu Tyr Leu Val Phe 65 70 75 80 Glu Phe Leu His Gln Asp Leu Lys Lys Phe Met Asp Ala Ser Ala Val 85 90 95 Thr Gly Ile Pro Leu Pro Leu Ile Lys Ser Tyr Leu Phe Gln Leu Leu 100 105 110 Gln Gly Leu Ala Phe Cys His Ser His Arg Val Leu His Arg Asp Leu 115 120 125 Lys Pro Gln Asn Leu Leu Ile Asn Ala Glu Gly Ser Ile Lys Leu Ala 130 135 140 Asp Phe Gly Leu Ala Arg Ala Phe Gly Val Pro Val Arg Thr Tyr Thr 145 150 155 160 His Glu Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Ile Leu Leu Gly 165 170 175 Cys Lys Tyr Tyr Ser Thr Ala Val Asp Ile Trp Ser Leu Gly Cys Ile 180 185 190 Phe Ala Glu Met Val Thr Arg Arg Ala Leu Phe Pro Gly Asp Ser Glu 195 200 205 Ile Asp Gln Leu Phe Arg Ile Phe Arg Thr Leu Gly Thr Pro Asp Glu 210 215 220 Val Val Trp Pro Gly Val Thr Ser Met Pro Asp Tyr Lys Pro Ser Phe 225 230 235 240 Pro Lys Trp Ala Arg Gln Asp Phe Ser Lys Val Val Pro Pro Leu Asp 245 250 255 Glu Asp Gly Arg Ser Leu Leu Ser Gln Met Leu His Tyr Asp Pro Asn 260 265 270 Lys Arg Ile Ser Ala Lys Ala Ala Leu Ala His Pro Phe Phe Gln Asp 275 280 285 Val Thr Lys Pro Val Pro His Leu Arg Leu 290 295 40 2213 DNA Cricetulus griseus 40 attgtcaagg gctgagcttt gcttgcgttc catccagagc tggggctgcg gtgctcacct 60 tggcccccag gtcctccgcc ccgagtgttg ggcccgcttt cttcagggtt cccgggcccc 120 gctcccgggg gcctgagccg ccgcactagt gctccatgga gaacttccaa aaggtggaga 180 agatcggaga gggcacgtac ggagtggtgt acaaagccaa aaacaagttg acgggagaag 240 tggtggcgct taagaaaatc cggctcgaca ctgaaactga aggtgtgccc agtactgcca 300 tccgagagat ctctctgctt aaggaactta atcaccctaa tattgtcaag ctgctagatg 360 tcatccacac ggaaaataag ctttacttgg tttttgaatt tctgcaccag gacctcaaga 420 aatttatgga tgcctctgct gtcactggca ttcctcttcc cctcatcaag agctatttgt 480 tccagctgct ccagggccta gcattctgcc attctcaccg ggtcctgcac cgagacctta 540 agccccagaa tctgcttatc aatgcagagg ggtccatcaa gttggcagac tttggactag 600 caagagcctt tggagtccct gttcgtactt atactcatga ggtggtgacc ctgtggtacc 660 gagcacctga aatccttcta ggctgcaagt actactccac agccgtggat atctggagtc 720 tgggctgcat ctttgctgaa atggtgaccc ggcgggccct gtttcccgga gattctgaga 780 tcgaccaact cttccggatc ttccgaactc tggggacccc tgatgaggtg gtttggccgg 840 gagtcacttc tatgcctgat tataagccga gtttcccgaa gtgggctcgg caagatttta 900 gcaaagttgt gcctccactg gatgaagatg ggcggagctt gttatcgcaa atgctgcact 960 atgaccccaa caagcggatt tcagccaaag cagccctggc ccaccctttc ttccaggatg 1020 tgactaagcc agtaccccac cttcggctgt gatgtccttc ccaaagcctt cctcaccctt 1080 ggtctgactt gactctgggc cttttggaca caggtcagcc ttctactctt ttctggctgt 1140 cttagcactc actttttctc ttggccagcc agttcccggg actcagaggt gcagggaaag 1200 atgaggtgac aggaaaaagt tttggtatca gatgcactta acccagcttc cactaccttc 1260 tctccctctt agtcatcact gagggttggt atgcaaaaca aaacaaaact gaccattctc 1320 tacccacccc catgtcaagt ttggccatcc cagtcttata aattattgtg tcccatgttt 1380 aggaagacac accccagacc tcctggtgcc actgttttat aaaggccaac tgataagttg 1440 ggacatttag gaaccaagcc acagaggtct attttaatga attagatgaa aaaaaaaata 1500 gatccaaaca gtttatacct tagttgtagt gtctggcctc gcccgacaat caaggaggcc 1560 cagctggaga agcagaagaa atgttccctt tcagcactct gtctaccttc tgcagggcaa 1620 gaagtcagga gggggatagg gattggtctg ttcaccataa ttcacaaggc aaggtgaaag 1680 acactggaat ctttctttta gtaattttgt tttctggtgc taacgactga acacaggacc 1740 tcgccctcac taagcaaatg caccacccgt atgccagatt ttctagctcc tctttttggc 1800 gttttagctc ttcagtttgt cagaggtccc tgttgccatc ttcttcctct gagagtcacc 1860 cctgactcca tagtggaagt gaagctttag atgtcacttt gaaaggcttt gacctggtca 1920 ggtcatcggg ggacattttc tataaaagag ttaacaatta tatttatatt tcaagttata 1980 gtagtttgga tattttagtg ttttgatttt tgtttttttg gggggcgggg cagtgtggat 2040 ggatttgttg ccatgtgcac tttggaattt tgtaatgcaa ttgttgaaag aaaatatttc 2100 ttttatgatc ttctccctct accccttgtc ctccagtgtt ctgttaatat ttatttgtaa 2160 tttagtttgt aattcattaa aagaaattat tctcaaaaaa aaaaaaaaaa aaa 2213 41 346 PRT Cricetulus griseus 41 Met Glu Asn Phe Gln Lys Val Glu Lys Ile Gly Glu Gly Thr Tyr Gly 1 5 10 15 Val Val Tyr Lys Ala Lys Asn Lys Leu Thr Gly Glu Val Val Ala Leu 20 25 30 Lys Lys Ile Arg Leu Asp Thr Glu Thr Glu Gly Val Pro Ser Thr Ala 35 40 45 Ile Arg Glu Ile Ser Leu Leu Lys Glu Leu Asn His Pro Asn Ile Val 50 55 60 Lys Leu Leu Asp Val Ile His Thr Glu Asn Lys Leu Tyr Leu Val Phe 65 70 75 80 Glu Phe Leu His Gln Asp Leu Lys Lys Phe Met Asp Ala Ser Ala Val 85 90 95 Thr Gly Ile Pro Leu Pro Leu Ile Lys Ser Tyr Leu Phe Gln Leu Leu 100 105 110 Gln Gly Leu Ala Phe Cys His Ser His Arg Val Leu His Arg Asp Leu 115 120 125 Lys Pro Gln Asn Leu Leu Ile Asn Ala Glu Gly Ser Ile Lys Leu Ala 130 135 140 Asp Phe Gly Leu Ala Arg Ala Phe Gly Val Pro Val Arg Thr Tyr Thr 145 150 155 160 His Glu Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Ile Leu Leu Gly 165 170 175 Cys Lys Tyr Tyr Ser Thr Ala Val Asp Ile Trp Ser Leu Gly Cys Ile 180 185 190 Phe Ala Glu Met His Leu Val Cys Thr Gln His His Ala Lys Cys Cys 195 200 205 Gly Glu His Arg Arg Asn Gly Arg His Ser Leu Cys Pro Leu Cys Ser 210 215 220 Tyr Leu Glu Val Ala Ala Ser Gln Gly Gly Gly Met Thr Ala Val Ser 225 230 235 240 Thr Pro Tyr Pro Val Thr Arg Arg Ala Leu Phe Pro Gly Asp Ser Glu 245 250 255 Ile Asp Gln Leu Phe Arg Ile Phe Arg Thr Leu Gly Thr Pro Asp Glu 260 265 270 Val Val Trp Pro Gly Val Thr Ser Met Pro Asp Tyr Lys Pro Ser Phe 275 280 285 Pro Lys Trp Ala Arg Gln Asp Phe Ser Lys Val Val Pro Pro Leu Asp 290 295 300 Glu Asp Gly Arg Ser Leu Leu Ser Gln Met Leu His Tyr Asp Pro Asn 305 310 315 320 Lys Arg Ile Ser Ala Lys Ala Ala Leu Ala His Pro Phe Phe Gln Asp 325 330 335 Val Thr Lys Pro Val Pro His Leu Arg Leu 340 345 42 1041 DNA Cricetulus griseus 42 atggagaact tccaaaaggt ggagaagatc ggagagggca cgtacggagt ggtgtacaaa 60 gccaaaaaca agttgacggg agaagtggtg gcgcttaaga aaatccggct cgacactgaa 120 actgaaggtg tgcccagtac tgccatccga gagatctctc tgcttaagga acttaatcac 180 cctaatattg tcaagctgct agatgtcatc cacacggaaa ataagcttta cttggttttt 240 gaatttctgc accaggacct caagaaattt atggatgcct ctgctgtcac tggcattcct 300 cttcccctca tcaagagcta tttgttccag ctgctccagg gcctagcatt ctgccattct 360 caccgggtcc tgcaccgaga ccttaagccc cagaatctgc ttatcaatgc agaggggtcc 420 atcaagttgg cagactttgg actagcaaga gcctttggag tccctgttcg tacttatact 480 catgaggtgg tgaccctgtg gtaccgagca cctgaaatcc ttctaggctg caagtactac 540 tccacagccg tggatatctg gagtctgggc tgcatctttg ctgaaatgca cctagtgtgt 600 acccagcacc atgctaagtg ctgtggggaa cacagaagaa atggaagaca cagtctctgc 660 ccgctgtgct cctatctaga agtggctgcg tcacaaggag gggggatgac cgcagtgtct 720 accccatacc ccgtgacccg gcgggccctg tttcccggag attctgagat cgaccaactc 780 ttccggatct tccgaactct ggggacccct gatgaggtgg tttggccggg agtcacttct 840 atgcctgatt ataagccgag tttcccgaag tgggctcggc aagattttag caaagttgtg 900 cctccactgg atgaagatgg gcggagcttg ttatcgcaaa tgctgcacta tgaccccaac 960 aagcggattt cagccaaagc agccctggcc caccctttct tccaggatgt gactaagcca 1020 gtaccccacc ttcggctgtg a 1041 43 298 PRT Rattus Norvegicus 43 Met Glu Asn Phe Gln Lys Val Glu Lys Ile Gly Glu Gly Thr Tyr Gly 1 5 10 15 Val Val Tyr Lys Ala Lys Asn Lys Leu Thr Gly Glu Val Val Ala Leu 20 25 30 Lys Lys Ile Arg Leu Asp Thr Glu Thr Glu Gly Val Pro Ser Thr Ala 35 40 45 Ile Arg Glu Ile Ser Leu Leu Lys Glu Leu Asn His Pro Asn Ile Val 50 55 60 Lys Leu Leu Asp Val Ile His Thr Glu Asn Lys Leu Tyr Leu Val Phe 65 70 75 80 Glu Phe Leu His Gln Asp Leu Lys Lys Phe Met Asp Ala Ser Ala Leu 85 90 95 Thr Gly Leu Pro Leu Pro Leu Ile Lys Ser Tyr Leu Phe Gln Leu Leu 100 105 110 Gln Gly Leu Ala Phe Cys His Ser His Arg Val Leu His Arg Asp Leu 115 120 125 Lys Pro Gln Asn Leu Leu Ile Asn Ala Glu Gly Ser Ile Lys Leu Ala 130 135 140 Asp Phe Gly Leu Ala Arg Ala Phe Gly Val Pro Val Arg Thr Tyr Thr 145 150 155 160 His Glu Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Ile Leu Leu Gly 165 170 175 Cys Lys Tyr Tyr Ser Thr Ala Val Asp Ile Trp Ser Leu Gly Cys Ile 180 185 190 Phe Ala Glu Met Val Thr Arg Arg Ala Leu Phe Pro Gly Asp Ser Glu 195 200 205 Ile Asp Gln Leu Phe Arg Ile Phe Arg Thr Leu Gly Thr Pro Asp Glu 210 215 220 Val Val Trp Pro Gly Val Thr Ser Met Pro Asp Tyr Lys Pro Ser Phe 225 230 235 240 Pro Lys Trp Ala Arg Gln Asp Phe Ser Lys Val Val Pro Pro Leu Asp 245 250 255 Glu Asp Gly Arg Ser Leu Leu Ser Gln Met Leu His Tyr Asp Pro Asn 260 265 270 Lys Arg Ile Ser Ala Lys Ala Ala Leu Ala His Pro Phe Phe Gln Asp 275 280 285 Val Thr Lys Pro Val Pro His Leu Arg Leu 290 295 44 1400 DNA Rattus Norvegicus 44 ccggtttttt aaaagagact ttcttggcca ggtggaattt aatctcaccc aagggatggt 60 gaaaattgtg agggaaaggg ctgccgtgct cacccgggcc cccaggtcct ccgagtgtcc 120 ggcccgcttt ccgtcagggt tcccgggccc cgctcccggg ggcctgagcc gccctactag 180 cgctccatgg agaacttcca aaaggtggag aagattggag agggcacgta cggagtggtg 240 tacaaagcca aaaacaagtt gacgggagaa gttgtggcgc ttaagaaaat ccggctcgac 300 actgagactg aaggtgtgcc cagtactgcc atccgagaga tctccctcct taaggagctc 360 aatcacccta acatcgtcaa gctgctggat gtcatccaca cagaaaataa gctttatctg 420 gtctttgaat tcctgcacca ggacctcaag aagtttatgg atgcttctgc tctcactgga 480 cttcctcttc ccctcatcaa gagctatctg ttccagctgc tccagggcct agcattctgc 540 cattctcacc gtgtcctgca ccgagacctt aagccccaga acctgcttat caacgcagag 600 gggtccatca agctggctga ctttggacta gcaagagcct ttggagtccc tgtccgtact 660 tacactcatg aggtggtgac cctctggtac cgagcaccgg agattcttct gggctgcaag 720 tactactcca cagccgtgga catctggagc ctgggctgca tctttgccga aatggtgacc 780 cgcagggccc tattccctgg agactctgag attgaccaac tcttccggat ctttcggact 840 ctggggaccc cagatgaggt ggtttggcca ggagttactt ctatgcctga ttataagcca 900 agtttcccca agtgggctcg gcaggatttt agcaaggttg tgcctcccct ggatgaagac 960 ggacggagct tgttatctca aatgctgcac tatgacccca acaagcggat ttcagccaaa 1020 gcagccctgg ctcacccttt cttccaggat gtgactaaac cagtgcccca ccttcgactc 1080 tgatgtcctt cccaaagccc tcttcacctg tggtctgacc tgatcctggg ccttttggac 1140 acaggtcagc cttctgctgt tttctggctg tcttagcctt ttctctccgg ttctggggat 1200 tcagaggtgc agagaaggat taggtgaaaa gggagaaagt tttggcatta gatgcactta 1260 acccgacttc cagcttctct cctcttccca gtcatcactg agaaggatta gtattgaagc 1320 aaactgcaag tctccagtgt cagattgcat cactagctgc tatagtacgg taggatgcag 1380 tctgtgcatg ttttaactat 1400 45 346 PRT Rattus Norvegicus 45 Met Glu Asn Phe Gln Lys Val Glu Lys Ile Gly Glu Gly Thr Tyr Gly 1 5 10 15 Val Val Tyr Lys Ala Lys Asn Lys Leu Thr Gly Glu Val Val Ala Leu 20 25 30 Lys Lys Ile Arg Leu Asp Thr Glu Thr Glu Gly Val Pro Ser Thr Ala 35 40 45 Ile Arg Glu Ile Ser Leu Leu Lys Glu Leu Asn His Pro Asn Ile Val 50 55 60 Lys Leu Leu Asp Val Ile His Thr Glu Asn Lys Leu Tyr Leu Val Phe 65 70 75 80 Glu Phe Leu His Gln Asp Leu Lys Lys Phe Met Asp Ala Ser Ala Leu 85 90 95 Thr Gly Leu Pro Leu Pro Leu Ile Lys Ser Tyr Leu Phe Gln Leu Leu 100 105 110 Gln Gly Leu Ala Phe Cys His Ser His Arg Val Leu His Arg Asp Leu 115 120 125 Lys Pro Gln Asn Leu Leu Ile Asn Ala Glu Gly Ser Ile Lys Leu Ala 130 135 140 Asp Phe Gly Leu Ala Arg Ala Phe Gly Val Pro Val Arg Thr Tyr Thr 145 150 155 160 His Glu Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Ile Leu Leu Gly 165 170 175 Cys Lys Tyr Tyr Ser Thr Ala Val Asp Ile Trp Ser Leu Gly Cys Ile 180 185 190 Phe Ala Glu Met His Leu Val Cys Thr Gln His His Ala Lys Cys Cys 195 200 205 Gly Glu His Arg Arg Asn Gly Arg His Ser Leu Cys Pro Leu Cys Ser 210 215 220 Tyr Leu Glu Val Ala Ala Ser Gln Gly Gly Gly Met Thr Ala Val Ser 225 230 235 240 Thr Pro Tyr Pro Val Thr Arg Arg Ala Leu Phe Pro Gly Asp Ser Glu 245 250 255 Ile Asp Gln Leu Phe Arg Ile Phe Arg Thr Leu Gly Thr Pro Asp Glu 260 265 270 Val Val Trp Pro Gly Val Thr Ser Met Pro Asp Tyr Lys Pro Ser Phe 275 280 285 Pro Lys Trp Ala Arg Gln Asp Phe Ser Lys Val Val Pro Pro Leu Asp 290 295 300 Glu Asp Gly Arg Ser Leu Leu Ser Gln Met Leu His Tyr Asp Pro Asn 305 310 315 320 Lys Arg Ile Ser Ala Lys Ala Ala Leu Ala His Pro Phe Phe Gln Asp 325 330 335 Val Thr Lys Pro Val Pro His Leu Arg Leu 340 345 46 1544 DNA Rattus Norvegicus 46 ccggtttttt aaaagagact ttcttggcca ggtggaattt aatctcaccc aagggatggt 60 gaaaattgtg agggaaaggg ctgccgtgct cacccgggcc cccaggtcct ccgagtgtcc 120 ggcccgcttt ccgtcagggt tcccgggccc cgctcccggg ggcctgagcc gccctactag 180 cgctccatgg agaacttcca aaaggtggag aagattggag agggcacgta cggagtggtg 240 tacaaagcca aaaacaagtt gacgggagaa gttgtggcgc ttaagaaaat ccggctcgac 300 actgagactg aaggtgtgcc cagtactgcc atccgagaga tctccctcct taaggagctc 360 aatcacccta acatcgtcaa gctgctggat gtcatccaca cagaaaataa gctttatctg 420 gtctttgaat tcctgcacca ggacctcaag aagtttatgg atgcttctgc tctcactgga 480 cttcctcttc ccctcatcaa gagctatctg ttccagctgc tccagggcct agcattctgc 540 cattctcacc gtgtcctgca ccgagacctt aagccccaga acctgcttat caacgcagag 600 gggtccatca agctggctga ctttggacta gcaagagcct ttggagtccc tgtccgtact 660 tacactcatg aggtggtgac cctctggtac cgagcaccgg agattcttct gggctgcaag 720 tactactcca cagccgtgga catctggagc ctgggctgca tctttgccga aatgcaccta 780 gtgtgtaccc agcaccatgc taagtgctgt ggggagcaca gaagaaatgg aagacacagt 840 ctctgcccgc tgtgctccta tctagaagtg gctgcatcac aaggaggggg gatgaccgca 900 gtgtctaccc cataccccgt gacccgcagg gccctattcc ctggagactc tgagattgac 960 caactcttcc ggatctttcg gactctgggg accccagatg aggtggtttg gccaggagtt 1020 acttctatgc ctgattataa gccaagtttc cccaagtggg ctcggcagga ttttagcaag 1080 gttgtgcctc ccctggatga agacggacgg agcttgttat ctcaaatgct gcactatgac 1140 cccaacaagc ggatttcagc caaagcagcc ctggctcacc ctttcttcca ggatgtgact 1200 aaaccagtgc cccaccttcg actctgatgt ccttcccaaa gccctcttca cctgtggtct 1260 gacctgatcc tgggcctttt ggacacaggt cagccttctg ctgttttctg gctgtcttag 1320 ccttttctct ccggttctgg ggattcagag gtgcagagaa ggattaggtg aaaagggaga 1380 aagttttggc attagatgca cttaacccga cttccagctt ctctcctctt cccagtcatc 1440 actgagaagg attagtattg aagcaaactg caagtctcca gtgtcagatt gcatcactag 1500 ctgctatagt acggtaggat gcagtctgtg catgttttaa ctat 1544 47 298 PRT Carassius auratus 47 Met Glu Ser Phe Gln Lys Val Glu Lys Ile Gly Glu Gly Thr Tyr Gly 1 5 10 15 Val Val Tyr Lys Ala Lys Asn Lys Val Thr Gly Glu Thr Val Ala Leu 20 25 30 Lys Lys Ile Arg Leu Asp Thr Glu Thr Glu Gly Val Pro Ser Thr Ala 35 40 45 Ile Arg Glu Ile Ser Leu Leu Lys Glu Leu Asn His Pro Asn Ile Val 50 55 60 Lys Leu His Asp Val Ile His Thr Glu Asn Lys Leu Tyr Leu Val Phe 65 70 75 80 Glu Phe Leu His Gln Asp Leu Lys Arg Phe Met Asp Ser Ser Thr Val 85 90 95 Thr Gly Ile Ser Leu Pro Leu Val Lys Ser Tyr Leu Phe Gln Leu Leu 100 105 110 Gln Gly Leu Ala Phe Cys His Ser His Arg Val Leu His Arg Asp Leu 115 120 125 Lys Pro Gln Asn Leu Leu Ile Asn Ala Gln Gly Glu Ile Lys Leu Ala 130 135 140 Asp Phe Gly Leu Ala Arg Ala Phe Gly Val Pro Val Arg Thr Tyr Thr 145 150 155 160 His Glu Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Ile Leu Leu Gly 165 170 175 Cys Lys Tyr Tyr Ser Thr Ala Val Asp Ile Trp Ser Leu Gly Cys Ile 180 185 190 Phe Ala Glu Met Ile Thr Arg Lys Ala Leu Phe Pro Gly Asp Ser Glu 195 200 205 Ile Asp Gln Leu Phe Arg Ile Phe Arg Thr Leu Gly Thr Pro Asp Glu 210 215 220 Ser Ile Trp Pro Gly Val Thr Ser Met Pro Asp Tyr Lys Pro Ser Phe 225 230 235 240 Pro Lys Trp Ala Arg Gln Asp Leu Ser Lys Val Val Pro Pro Leu Asp 245 250 255 Glu Asp Gly Arg Asp Leu Leu Gly Gln Met Leu Ile Tyr Asp Pro Asn 260 265 270 Lys Arg Ile Ser Ala Lys Asn Ala Leu Val His Arg Phe Phe Arg Asp 275 280 285 Val Thr Met Pro Val Pro Pro Leu Arg Leu 290 295 48 1205 DNA Carassius auratus 48 ctaaaattac tctctctata cagccgctgt tttaagtagc tttagagtgg cagttttccc 60 cggaaaaggg gcagtttgac atggagtcct ttcagaaagt cgagaagatt ggagaaggaa 120 catacggggt tgtttataaa gccaagaata aagtcaccgg agagacagtt gcactaaaga 180 aaattcgatt agacacagag actgaaggtg ttcccagcac tgccatacgt gagatctctc 240 tgctaaaaga gctcaatcac ccaaacatag tcaagttgca tgatgtgata cacacagaaa 300 ataagcttta cttggtcttt gaatttcttc accaagacct gaagaggttt atggactcgt 360 ccactgtcac tggcatatcc ttgccactcg tgaagagtta cctgttccag ttgctccagg 420 gactggcctt ctgtcactct catcgtgttc ttcataggga tcttaaaccc cagaatctcc 480 tgatcaacgc tcagggtgag atcaaactgg ctgactttgg tctggccaga gcgtttggtg 540 tacctgtgcg gacttacaca cacgaggttg taactttgtg gtacagagct ccagagattc 600 tcctgggatg taaatattat tctacagcgg ttgacatctg gagtttgggc tgtatctttg 660 cagaaatgat cactcggaag gctttgtttc ctggagactc tgaaatagac cagctctttc 720 ggatatttcg gacacttggc actccggatg aatctatatg gcctggagtc acctcaatgc 780 cagactacaa accctccttt cccaagtggg cacgacagga cctgtctaaa gtggtgccac 840 ccctggatga agatggcaga gaccttcttg ggcaaatgtt gatctatgat cctaataaga 900 ggatctcagc aaagaacgcc cttgttcatc ggttcttccg tgatgtcacc atgccagtgc 960 cccccttgcg cctctgaagt catccgtgat gatccttcac agcactcgag tttggatcca 1020 ctttctgaga gttattcccc atttctttgg aaattgatca gttttatact atgacttttt 1080 ttctccatta ttcaaagata atatggttct aattaatgat tttaaaccaa atttctgatg 1140 gattcataaa atagtggtct ctgcaactta ataaacttgt tacattgtta aaaaaaaaaa 1200 aaaaa 1205 49 299 PRT Sphaerechinus granularis 49 Met Asn Asn Phe Glu Lys Ile Glu Lys Ile Gly Glu Gly Thr Tyr Gly 1 5 10 15 Val Val Tyr Lys Ala Lys Asp Leu Lys Ser Gly Lys Thr Val Ala Leu 20 25 30 Lys Lys Ile Arg Leu Asp Thr Glu Ser Glu Gly Val Pro Ser Thr Ala 35 40 45 Ile Arg Glu Ile Ala Leu Leu Lys Glu Leu Asp His Lys Asn Ile Val 50 55 60 Lys Leu His Asp Val Val His Ser Asp Lys Lys Leu Tyr Leu Val Phe 65 70 75 80 Glu Phe Met Asn Gln Asp Leu Lys Lys Tyr Met Asp Ile Ala Pro Pro 85 90 95 Ser Gly Leu Pro Thr Ala Leu Val Lys Ser Tyr Leu Gln Gln Leu Leu 100 105 110 His Gly Ile Ala Phe Cys His Ala His Arg Val Leu His Arg Asp Leu 115 120 125 Lys Pro Gln Asn Leu Leu Ile Asp Ala Asp Gly His Ile Lys Leu Ala 130 135 140 Asp Phe Gly Leu Ala Arg Ala Phe Gly Val Pro Val Arg Thr Tyr Thr 145 150 155 160 His Glu Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Ile Leu Leu Gly 165 170 175 Cys Arg Phe Tyr Ser Thr Ala Val Asp Ile Trp Ser Ile Gly Cys Ile 180 185 190 Phe Val Glu Met Ile Thr Arg Arg Ala Leu Phe Pro Gly Asp Ser Glu 195 200 205 Ile Asp Gln Leu Phe Arg Ile Phe Arg Thr Met Gly Thr Pro Asp Glu 210 215 220 Lys Leu Trp Pro Gly Val Thr Ser Leu Pro Asp Tyr Lys Thr Ser Phe 225 230 235 240 Pro Arg Trp Ser Pro Gln Asp Phe Asn Lys Ile Val Pro Met Leu Ser 245 250 255 Lys Asp Gly Lys Asp Leu Leu Lys Cys Met Leu Cys Tyr Glu Pro Asp 260 265 270 Lys Arg Ile Ser Ala Lys Thr Ala Leu Ser His Pro Tyr Phe Lys Asp 275 280 285 Val Lys Leu Val Pro Pro Pro His Leu Pro Thr 290 295 50 1410 DNA Sphaerechinus granularis 50 attatcacta cggtatggcg ttagaagacc gtcaatatga ataactttga aaagattgag 60 aagattggcg aaggcactta tggagtggtt tacaaggcaa aagacctaaa atctggaaaa 120 accgtcgcac tgaagaaaat acgattggat acagaatcgg aaggtgtccc aagtactgcc 180 atcagagaaa tcgcactctt gaaggaactg gaccacaaga atatagtaaa attacatgat 240 gtggtgcaca gtgataagaa gctgtatctt gtctttgagt tcatgaacca ggatctcaag 300 aaatacatgg atattgcacc tccgtctggt ctaccaacag ctttagtcaa gagttatctt 360 cagcaacttc tacatggtat agcattctgc catgcacacc gtgtccttca cagagacctc 420 aagcctcaga atctgctcat tgacgcagat ggccacatca aacttgctga ttttggacta 480 gcaagagcat ttggggttcc tgtcaggaca tacactcatg aggttgtcac attatggtat 540 cgtgctcccg aaatcttact aggctgcagg ttttactcta cagcggtgga catctggagt 600 ataggatgta tctttgtaga gatgataaca agaagagctc tattccctgg tgattctgaa 660 attgaccagc tcttcaggat attccgaaca atgggcactc cagatgaaaa actctggcct 720 ggggtcacct ctctacccga ctacaaaacc agtttcccaa gatggtcacc tcaggatttc 780 aacaagatag tccccatgct cagtaaagat ggcaaggact tactcaagtg tatgctgtgc 840 tacgaaccag acaagaggat atcagccaag acagctctct cacatccata tttcaaggat 900 gttaaactgg tgccgcctcc tcatcttcca acatgatcaa cttgcaccat gcaaagcctt 960 cttatagata aaatgtatgt gttgaagaga gacattacct tatttattac acatctttgt 1020 gtatattatg tataaatagt gagaaagggt ttgtgttttg tttatggtta ttttgtggtg 1080 tacatgacaa tattccactt agcttactag taatctgatt gctctaggac tgtaataatt 1140 gaacattata caacaatttt gtgccttaaa aattatgtat gtggggtgac aacgcattat 1200 atctcaaatc tattgacttt tcaggaggat aaaaagacat ggtcattatg aaagtactct 1260 ctgtcatgat atccttcatt tacagagcaa tagtgacttg aactctgcag tgcgtatttt 1320 gaggaataag ggatttgaca agacaaattt tgtctcgagc tattaagcat gaaaaacccc 1380 accaggttga gctactgggt ttcacacctg 1410 51 297 PRT Xenopus laevis 51 Met Glu Asn Phe Gln Lys Val Glu Lys Ile Gly Glu Gly Thr Tyr Gly 1 5 10 15 Val Val Tyr Lys Ala Arg Asn Arg Glu Thr Gly Glu Ile Val Ala Leu 20 25 30 Lys Lys Ile Arg Leu Asp Thr Glu Thr Glu Gly Val Pro Ser Thr Ala 35 40 45 Ile Arg Glu Ile Ser Leu Leu Lys Glu Leu Asn His Pro Asn Ile Val 50 55 60 Lys Leu Leu Asp Val Ile His Thr Glu Asn Lys Leu Tyr Leu Val Phe 65 70 75 80 Glu Phe Leu Asn Gln Asp Leu Lys Lys Phe Met Asp Gly Ser Asn Ile 85 90 95 Ser Gly Ile Ser Leu Ala Leu Val Lys Ser Tyr Leu Phe Gln Leu Leu 100 105 110 Gln Gly Leu Ala Phe Cys His Ser His Arg Val Leu His Arg Asp Leu 115 120 125 Lys Pro Gln Asn Leu Leu Ile Asn Ser Asp Gly Ala Ile Lys Leu Ala 130 135 140 Asp Phe Gly Leu Ala Arg Ala Phe Gly Val Pro Val Arg Thr Phe Thr 145 150 155 160 His Glu Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Ile Leu Leu Gly 165 170 175 Cys Lys Phe Tyr Ser Thr Ala Val Asp Ile Trp Ser Leu Gly Cys Ile 180 185 190 Phe Ala Glu Met Ile Thr Arg Arg Ala Leu Phe Pro Gly Asp Ser Glu 195 200 205 Ile Asp Gln Leu Phe Arg Ile Phe Arg Thr Leu Gly Thr Pro Asp Glu 210 215 220 Val Ser Trp Pro Gly Val Thr Thr Met Pro Asp Tyr Lys Ser Thr Phe 225 230 235 240 Pro Lys Trp Ile Arg Gln Asp Phe Ser Lys Val Val Pro Pro Leu Asp 245 250 255 Glu Asp Gly Arg Asp Leu Leu Ala Gln Met Leu Gln Tyr Asp Ser Asn 260 265 270 Lys Arg Ile Ser Ala Lys Val Ala Leu Thr His Pro Phe Phe Arg Asp 275 280 285 Val Ser Arg Pro Thr Pro His Leu Ile 290 295 52 1131 PRT Xenopus laevis 52 Ile Pro Arg Gly Thr Lys Pro Leu Pro Ser Pro Val Ser Phe Leu Gln 1 5 10 15 Leu Ser Thr Gln Arg Glu Val Val Ala Arg Val Ile Gln Arg Ile Cys 20 25 30 Glu Lys Lys Arg Lys Asn Val Leu Ala Phe Gly Tyr Gly Leu Val Asp 35 40 45 Glu Lys Asn Ser Leu Asn Ile Arg Leu Thr Pro Asn Ile Cys Asn Tyr 50 55 60 Phe Pro Asn Pro Thr Thr Thr Thr Ile Ser Thr Ser Ile Leu Trp Glu 65 70 75 80 Thr Leu Leu Thr Arg Val Gly Asp Asp Val Met Met Tyr Trp Leu Glu 85 90 95 Gln Cys Ser Ile Phe Val Phe Val Pro Pro Arg Cys Cys Tyr Gln Ile 100 105 110 Thr Gly Gln Pro Ile Tyr Thr Leu Pro Ser Asp Asp Val Phe Leu Phe 115 120 125 Gln Ser Gln Ser Phe Thr Gln Ser Asn Val Leu Leu Arg Tyr Ile Lys 130 135 140 Arg Asn Val Phe His Leu Arg Lys Lys Tyr Leu Lys Pro Lys His Ser 145 150 155 160 Met Thr Ser Arg Met Leu Thr Trp Arg Arg Asn Lys Ser Pro Ser Gly 165 170 175 Leu Leu Ile Arg Ser Lys Thr Ser Met Ala Val Thr Thr Glu Ile His 180 185 190 Ser Lys Arg Lys Leu Cys Ser Lys Asp Ile Cys Val Ile Pro Asp Lys 195 200 205 Lys Arg Arg Asp Asn Leu Asp Lys Asp Asp Thr Val Asp His Phe Asp 210 215 220 Leu Pro Met Cys Arg Ser Val Ser Tyr Leu Ser Asn Met Tyr Pro Lys 225 230 235 240 Thr Asn Val Gln Val Thr Gly Leu Ile Thr Ser Gly Tyr Lys Lys Thr 245 250 255 Lys Thr Phe Gln Cys Gln Lys Pro Val Ser Cys Glu Gln Lys Lys Thr 260 265 270 Thr Ala Phe Tyr Ser Val Ala Gly Asp Cys Asn Leu Ser Leu Lys Asp 275 280 285 Asn Val Asn Lys Leu Ile Thr Asn Ala Ser Val Pro Thr Ala Gln Ser 290 295 300 Arg Leu Ser Phe Ser Asn Ile Phe Ile Asp Phe Gly Arg Thr Leu Tyr 305 310 315 320 Leu Ser Ile Ser Tyr Lys Lys Gly Phe Ser Glu Ser Phe Ile Leu Asn 325 330 335 Ser Leu Asp Ser Thr Pro Ser Gly Ser Gln Lys Leu Val Glu Thr Ile 340 345 350 Phe Leu Ser Asn Phe Leu Ala Glu Gln Asn Phe Asp Gln Pro Lys Arg 355 360 365 Asp Glu Asn Cys Arg Tyr Lys Leu Pro Lys Arg Tyr Trp Lys Met Lys 370 375 380 Pro His Phe Gln Glu Leu Ile Gln Asn His Lys Lys Phe Pro Tyr Leu 385 390 395 400 Val Tyr Leu Asn Lys His Cys Pro Val Arg Ser Ser Met Ala Cys Ser 405 410 415 Glu Lys Arg Ser Leu Gln Lys Asn Arg Ile Glu Asn Asp Gly Lys Gln 420 425 430 Leu Lys His Phe Thr Thr Lys Ala Asn Leu Leu Ser Leu Leu Lys Gln 435 440 445 His Ser Ser Ile Trp Gln Val Tyr Met Phe Val Arg Glu Cys Leu Asn 450 455 460 Asn Val Val Pro Asp Ile Met Trp Gly Ser Ser His Asn Lys Cys Arg 465 470 475 480 Phe Phe Arg Asn Val Lys Ser Phe Leu Phe Phe Ser Gly Lys Phe Gly 485 490 495 Lys Ile Ser Leu Ser Glu Leu Met Trp Ser Met Arg Val Glu Asp Cys 500 505 510 Ser Trp Ile Arg Leu Gln Lys Ser Asp His Phe Val Pro Ala Ser Glu 515 520 525 His Leu Leu Arg Glu Lys Ile Leu Ala Lys Phe Val Phe Trp Leu Met 530 535 540 Asp Thr Tyr Val Ile Gln Leu Leu Lys Ser Phe Phe Tyr Val Thr Glu 545 550 555 560 Thr Met Phe Gln Lys His Arg Leu Leu Phe Tyr Arg Lys Ser Val Trp 565 570 575 Lys Lys Leu Gln Asn Ile Gly Leu Arg Lys His Leu Glu Lys Val Lys 580 585 590 Leu Arg Ser Leu Ser Ser Asp Glu Leu Glu Asn Met Gln Gln Trp Lys 595 600 605 Asn Val Pro Leu Val Ser Arg Leu Arg Phe Ile Pro Lys Thr Asn Gly 610 615 620 Leu Arg Pro Ile Ser Lys Ile Ser Ser Thr Leu Ser Ser Gln Gln Ser 625 630 635 640 Lys Glu Asn Gln Glu Lys Lys Ile His His Phe Ser Ser Gln Ile Arg 645 650 655 Asn Leu Phe Ser Val Leu Asn Tyr Glu Trp Asn Arg Asn Cys Ser Leu 660 665 670 Ile Gly Ser Ser Val Phe Gly Met Asp Asp Ile Tyr Lys Lys Trp Lys 675 680 685 Lys Phe Val Leu Asp Phe Glu Lys Pro Gln Val Glu Lys Leu Gln Phe 690 695 700 Tyr Phe Val Lys Thr Asp Val Lys Gly Ala Tyr Asp Thr Ile Pro His 705 710 715 720 Ser Lys Leu Asp Glu Val Ile Ser Lys Val Ile Asn Pro Asn Ala Asn 725 730 735 Glu Val Tyr Cys Ile Arg Arg Tyr Ala Thr Val Ser Val Asp Pro Thr 740 745 750 Gly Arg Ile Ile Lys Ser Phe Lys Arg His Val Ser Glu Leu Ala Asp 755 760 765 Val Leu Pro Asn Met Lys Gln Phe Val Ser Asn Gln Gln Glu Lys Asn 770 775 780 Leu Leu Arg Asn Thr Ile Leu Val Glu Gln Asn Leu Leu Leu Asn Glu 785 790 795 800 Ser Ser Val Lys Leu Leu Ala Val Phe Gln Gln Ile Ile Arg Ser His 805 810 815 Ile Leu Arg Ile Lys Asp Arg Tyr Tyr Met Gln Cys Cys Gly Ile Pro 820 825 830 Gln Gly Ser Met Leu Ser Thr Ile Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845 Met Glu Asn Ala Met Leu Gly Gly Ile Gln Lys Asn Gly Val Leu Met 850 855 860 Arg Leu Ile Asp Asp Phe Leu Leu Val Thr Pro His Leu Asp Gln Ala 865 870 875 880 Lys Thr Phe Leu Arg Thr Leu Ala Glu Gly Ile Pro Gln Tyr Gly Cys 885 890 895 Ser Ile Ser Pro Gln Lys Thr Val Val Asn Phe Pro Val Asp Asp Ile 900 905 910 Pro Glu Cys Ser Glu Val Glu Gln Leu Pro Ser His Cys Leu Phe Arg 915 920 925 Trp Cys Gly Leu Leu Leu Asp Thr Gln Thr Leu Asp Val Tyr Tyr Asp 930 935 940 Tyr Ser Ser Tyr Ala Cys Thr Ser Ile Arg Ser Ser Met Thr Phe Cys 945 950 955 960 His Ser Ser Ala Ala Gly Lys Tyr Met Lys Gln Lys Leu Ile Arg Val 965 970 975 Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Lys Val Asn Ser 980 985 990 Leu Arg Thr Val Cys Ile Asn Thr Tyr Lys Ile Phe Leu Leu Gln Ala 995 1000 1005 Tyr Arg Phe His Ala Cys Val Val Gln Leu Pro Phe Gly Gln Arg Val 1010 1015 1020 Met Asn Asn Pro Pro Phe Phe Leu Thr Val Ile Ser Asp Met Ala Pro 1025 1030 1035 1040 Cys Phe Tyr Thr Thr Phe Lys Ala Lys Asn Lys Asp Leu Thr Arg Gly 1045 1050 1055 Tyr Lys Asp Val Ser Cys Gln Phe Asn Phe Glu Ala Val Gln Trp Leu 1060 1065 1070 Ser Tyr Gln Ala Phe Leu Thr Lys Leu His Asn His Lys Val Leu Tyr 1075 1080 1085 Lys Cys Leu Ile Gly Pro Leu Gln Asn Cys Lys Met Gln Leu Ser Arg 1090 1095 1100 Arg Leu Ser Gln Asp Thr Ile Glu Leu Leu Lys Ser Val Thr Asp Ser 1105 1110 1115 1120 Ser Leu His Lys Asp Phe Ser Cys Ile Met Asp 1125 1130 53 1128 PRT Mesocricetus auratus 53 Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ala Leu Leu Arg Ser 1 5 10 15 Gln Tyr Arg Gln Val Trp Pro Leu Ala Thr Phe Val Arg Arg Leu Gly 20 25 30 Pro Glu Gly Arg Gln Leu Val Gln Pro Gly Asp Pro Lys Val Phe Arg 35 40 45 Thr Leu Val Ala Arg Cys Leu Val Cys Val Pro Trp Asp Ser Gln Pro 50 55 60 Pro Pro Ala Asp Leu Ser Phe His Gln Val Ser Ser Leu Lys Glu Leu 65 70 75 80 Val Ala Arg Val Val Gln Arg Leu Cys Glu Arg Gly Glu Arg Asn Val 85 90 95 Leu Thr Phe Gly Phe Ala Leu Leu Asn Gly Ala Gln Gly Gly Pro Pro 100 105 110 Met Thr Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Ser Val Thr 115 120 125 Glu Ser Leu Arg Val Ser Gly Ala Trp Met Leu Leu Leu Asn Arg Val 130 135 140 Gly Asp Asp Leu Leu Val Tyr Leu Leu Ala Arg Cys Ala Leu Tyr Leu 145 150 155 160 Leu Val Pro Pro Ser Cys Ala Tyr Gln Val Cys Gly Ser Pro Leu Tyr 165 170 175 Gln Ile Cys Ala Thr Ala Glu Thr Trp Pro Ser Val Ser Arg Ile Tyr 180 185 190 Arg Pro Thr Arg Pro Val Gly Arg Asn Phe Thr His Leu Gly Ser Thr 195 200 205 His Arg Val Arg Asn Ser Ser His Gln Glu Ala Trp Lys Pro Pro Pro 210 215 220 Leu Pro Ser Arg Glu Ala Lys Arg Ser Leu Ser Ile Thr Asn Arg Ser 225 230 235 240 Val Pro Pro Ser Lys Lys Ala Arg Cys Asp Leu Ala Pro Arg Leu Glu 245 250 255 Lys Gly Pro Tyr Arg Gln Ala Val Pro Thr Pro Ser Asp Lys Thr Trp 260 265 270 Val Pro Asn Pro Ala Lys Ser His Ala Val Pro Ile Ser Arg Thr Thr 275 280 285 Lys Glu Asp Leu Ser Ser Gly Val Lys Ala Pro Gly Leu Ser Arg Ser 290 295 300 Gly Ser Val Cys Tyr Lys His Lys Pro Ser Ser Thr Ser Leu Gln Ser 305 310 315 320 Pro Leu Cys Gln Asn Ala Phe Gln Leu Arg Pro Tyr Thr Glu Thr Lys 325 330 335 Arg Phe Leu Tyr Ser Arg Glu Gly Gly Arg Glu Arg Leu Asn Pro Ser 340 345 350 Phe Leu Leu Asn Asn Leu Gln Pro Ser Leu Thr Gly Ala Arg Arg Leu 355 360 365 Val Glu Ile Leu Phe Leu Gly Met Arg Pro Arg Thr Ser Gly Pro Leu 370 375 380 Cys Gly Arg Arg Arg Leu Ser Lys Arg Tyr Trp Gln Met Arg Pro Leu 385 390 395 400 Phe Gln Gln Leu Leu Val Asn His Ala Arg Cys Pro Tyr Val Arg Leu 405 410 415 Leu Arg Ser His Cys Arg Phe Arg Thr Ala Ala His Gln Val Ala Gly 420 425 430 Ala Leu Asn Thr Thr Ser Pro Gln Arg Leu Met Asn Leu Leu Arg Leu 435 440 445 His Ser Ser Pro Trp Gln Val Tyr Gly Phe Leu Gln Ala Cys Val Gly 450 455 460 Lys Leu Val Pro Pro Gly Leu Trp Gly Ser Arg His Asn Gln Arg Arg 465 470 475 480 Phe Phe Lys Asn Val Lys Arg Phe Ile Ser Leu Gly Lys Tyr Asp Lys 485 490 495 Leu Ser Leu Gln Glu Leu Thr Trp Lys Met Lys Val Gln Asp Cys Arg 500 505 510 Trp Leu Arg Ser Ser Pro Gly Asn Asn Cys Val Pro Ala Ala Glu His 515 520 525 Arg Thr Arg Glu Arg Ile Leu Ala Val Phe Leu Phe Trp Leu Met Asp 530 535 540 Ala Tyr Val Val Glu Leu Leu Arg Ser Phe Phe Tyr Val Thr Glu Thr 545 550 555 560 Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr Arg Lys Ser Met Trp Arg 565 570 575 Arg Leu Gln Ser Ile Gly Val Arg His His Leu Glu Arg Val Arg Leu 580 585 590 Gln Glu Leu Ser Gln Glu Glu Val Arg Gln Arg Gln Glu Ala Trp Pro 595 600 605 Ala Met Pro Ile Cys Arg Leu Arg Phe Ile Pro Lys Pro Ser Gly Leu 610 615 620 Arg Pro Ile Val Asn Met Ser Tyr Met Gly Thr Arg Ala Phe Asp Lys 625 630 635 640 Gly Lys Gln Ala Gln His Phe Thr Gln Cys Leu Lys Thr Leu Phe Ser 645 650 655 Val Leu Asn Tyr Glu Leu Thr Lys His Thr Asn Leu Leu Gly Ala Ser 660 665 670 Val Leu Gly Leu Asn Asp Ile Tyr Arg Thr Trp Arg Thr Phe Val Leu 675 680 685 Arg Val Arg Thr Leu Asp Pro Ala Pro Arg Met Tyr Phe Val Lys Ala 690 695 700 Asp Val Thr Gly Ala Tyr Asp Ala Ile Pro Gln Asp Lys Leu Val Glu 705 710 715 720 Val Ile Ala Asn Met Ile Arg His Pro Asp Asn Ser Tyr Cys Ile His 725 730 735 Gln Tyr Ala Val Val Gln Arg Asp Arg Gln Gly Gln Ile His Lys Ser 740 745 750 Phe Arg Arg Gln Val Ser Thr Leu Ser Asp Leu Gln Pro His Met Gly 755 760 765 Gln Phe Leu Lys His Leu Gln Asp Ser Asp Thr Ser Ala Leu Arg Asn 770 775 780 Ser Val Val Ile Glu Gln Ser Leu Ser Leu Asn Glu Ala Ser Ser Ser 785 790 795 800 Leu Phe Asp Phe Phe Leu Arg Phe Val Arg Asn Ser Val Val Lys Ile 805 810 815 Gly Gly Arg Cys Tyr Val Gln Cys Gln Gly Ile Pro Gln Gly Ser Ser 820 825 830 Leu Ser Thr Leu Leu Cys Ser Leu Cys Phe Gly Asp Met Glu Asn Lys 835 840 845 Leu Phe Ala Glu Val Gln Gln Asp Gly Leu Leu Leu Arg Phe Val Asp 850 855 860 Asp Phe Leu Leu Val Thr Pro His Leu Val Gln Ala Glu Ala Phe Leu 865 870 875 880 Arg Ala Leu Val Arg Gly Ile Pro Glu Tyr Gly Cys Met Ile Asn Leu 885 890 895 Gln Lys Thr Val Val Asn Phe Pro Val Asp Ala Gly Thr Leu Asp Gly 900 905 910 Thr Ala Pro His Gln Leu Pro Ala His Cys Leu Phe Pro Trp Cys Gly 915 920 925 Leu Leu Leu Asp Thr Gln Thr Leu Glu Val Leu Cys Asp Tyr Thr Gly 930 935 940 Tyr Ala Arg Thr Ser Ile Lys Ala Ser Leu Thr Phe Gln Arg Thr Phe 945 950 955 960 Lys Ala Gly Arg Asn Met Arg Gln Lys Leu Leu Ala Val Leu Arg Leu 965 970 975 Lys Cys His Ser Leu Phe Leu Asp Leu Gln Met Asn Ser Leu Gln Thr 980 985 990 Val Cys Ile Asn Val Tyr Lys Ile Phe Leu Leu Gln Ala Tyr Arg Phe 995 1000 1005 His Ala Cys Ala Leu Gln Leu Pro Phe Asp Gln His Val Arg Lys Asn 1010 1015 1020 Pro Ala Phe Phe Leu Ser Ile Ile Ser Asn Ile Ala Ser Cys Cys Tyr 1025 1030 1035 1040 Ser Ile Leu Lys Val Lys Asn Ala Gly Met Thr Leu Lys Ala Lys Gly 1045 1050 1055 Ala Ser Gly Ser Phe Pro Pro Glu Ala Ala Arg Trp Leu Cys Tyr Gln 1060 1065 1070 Ala Phe Leu Leu Lys Leu Ala Gly His Ser Val Thr Tyr Lys Cys Leu 1075 1080 1085 Leu Gly Pro Leu Arg Thr Ala Gln Lys Gln Leu Cys Arg Lys Leu Pro 1090 1095 1100 Arg Ala Thr Met Ala Ile Leu Glu Thr Ala Ala Asp Pro Ala Leu Ser 1105 1110 1115 1120 Thr Asp Phe Gln Thr Ile Leu Asp 1125 54 2629 PRT Rattus Norvegicus 54 Met Glu Lys Leu Cys Gly Tyr Val Pro Val His Pro Asp Ile Leu Ser 1 5 10 15 Leu Lys Asn Arg Cys Leu Thr Met Leu Ser Asp Ile Gln Pro Leu Glu 20 25 30 Lys Ile His Gly Gln Arg Ser Val Asn Pro Asp Ile Leu Ser Leu Glu 35 40 45 Asn Arg Cys Leu Thr Leu Leu Pro Asp Leu Gln Pro Met Glu Lys Ile 50 55 60 His Gly Gln Arg Ser Val His Pro Asp Ile Leu Ser Ser Glu Asn Arg 65 70 75 80 Cys Leu Thr Leu Leu Pro Asp Leu Gln Ser Leu Glu Lys Leu Cys Gly 85 90 95 His Met Ser Ser His Pro Asp Val Leu Ser Leu Glu Asn Arg Cys Leu 100 105 110 Ala Thr Leu Pro Thr Val Lys Arg Thr Val Ser Ser Gly Pro Leu Leu 115 120 125 Gln Cys Leu His Arg Ser His Thr Ala Gln Ala Asp Leu Arg Asp Pro 130 135 140 Asn Phe Arg Asn Cys Leu Phe Pro Glu Pro Pro Thr Ile Glu Ala Pro 145 150 155 160 Cys Phe Leu Lys Glu Leu Asp Leu Pro Thr Gly Pro Arg Ala Leu Lys 165 170 175 Ser Met Ser Ala Thr Ala Arg Val Gln Glu Val Ala Leu Gly Gln Arg 180 185 190 Cys Val Ser Glu Gly Lys Glu Leu Gln Glu Glu Lys Glu Ser Ala Glu 195 200 205 Val Pro Met Pro Leu Tyr Ser Leu Ser Leu Gly Gly Glu Glu Glu Glu 210 215 220 Val Val Gly Ala Pro Val Leu Lys Leu Thr Ser Gly Asp Ser Asp Ser 225 230 235 240 His Pro Glu Thr Thr Asp Gln Ile Leu Gln Glu Lys Lys Met Ala Leu 245 250 255 Leu Thr Leu Leu Cys Ser Ala Met Ala Ser Ser Val Asn Val Lys Asp 260 265 270 Ala Ser Asp Pro Thr Arg Ala Ser Ile His Glu Val Cys Ser Ala Leu 275 280 285 Ala Pro Leu Glu Pro Glu Phe Ile Leu Lys Ala Ser Leu Tyr Ala Arg 290 295 300 Gln Gln Leu Asn Leu Arg Asp Ile Ala Asn Ile Val Leu Ala Val Ala 305 310 315 320 Ala Leu Leu Pro Ala Cys Arg Pro His Val Arg Arg Tyr Tyr Ser Ala 325 330 335 Ile Val His Leu Pro Ser Asp Trp Ile Gln Val Ala Glu Phe Tyr Gln 340 345 350 Ser Leu Ala Glu Gly Asp Glu Lys Lys Leu Val Pro Leu Pro Ala Cys 355 360 365 Leu Arg Ala Ala Met Thr Asp Lys Phe Ala Gln Phe Asp Glu Tyr Gln 370 375 380 Leu Ala Lys Tyr Asn Pro Arg Lys His Arg Ser Lys Thr Arg Ser Arg 385 390 395 400 Gln Pro Pro Arg Pro Gln Arg Thr Lys Pro Pro Phe Ser Glu Ser Gly 405 410 415 Lys Cys Phe Pro Lys Ser Val Trp Pro Leu Lys Asn Glu Gln Ile Ser 420 425 430 Phe Glu Ala Ala Tyr Asn Ala Val Ser Glu Lys Lys Arg Leu Pro Arg 435 440 445 Phe Thr Leu Lys Lys Leu Val Glu Gln Leu His Ile His Glu Pro Ala 450 455 460 Gln His Val Gln Ala Leu Leu Gly Tyr Arg Tyr Pro Ser Thr Leu Glu 465 470 475 480 Leu Phe Ser Arg Ser His Leu Pro Gly Pro Trp Asp Ser Ser Arg Ala 485 490 495 Gly Gln Arg Met Lys Leu Gln Arg Pro Glu Thr Trp Glu Arg Glu Leu 500 505 510 Ser Leu Arg Gly Asn Arg Ala Ser Val Trp Glu Glu Leu Ile Asp Asn 515 520 525 Gly Lys Leu Pro Phe Met Ala Met Leu Arg Asn Leu Cys Asn Leu Leu 530 535 540 Arg Thr Gly Ile Ser Ala His His His Glu Leu Val Leu Gln Arg Leu 545 550 555 560 Gln His Glu Lys Ser Val Ile His Ser Arg Gln Phe Pro Phe Arg Phe 565 570 575 Leu Asn Ala His Asp Ser Leu Asp Arg Leu Glu Ala Gln Leu Arg Ser 580 585 590 Lys Ala Ser Pro Phe Pro Ser Asn Thr Thr Leu Met Lys Arg Ile Met 595 600 605 Ile Arg Asn Ser Lys Lys Ile Lys Arg Pro Ala Asn Pro Arg Tyr Leu 610 615 620 Cys Thr Leu Thr Gln Arg Gln Leu Arg Ala Ala Met Ala Ile Pro Val 625 630 635 640 Met Tyr Glu His Leu Lys Arg Glu Lys Leu Arg Leu His Lys Ala Arg 645 650 655 Gln Trp Thr Cys Asp Leu Glu Leu Leu Glu Arg Tyr Arg Gln Ala Leu 660 665 670 Glu Thr Ala Val Asn Ile Ser Val Lys His Asn Leu Pro Pro Leu Pro 675 680 685 Gly Arg Thr Leu Leu Val Tyr Leu Thr Asp Ala Asn Ala Asn Arg Leu 690 695 700 Cys Pro Lys Ser His Leu Gln Gly Pro Pro Leu Asn Tyr Val Leu Leu 705 710 715 720 Leu Ile Gly Met Met Met Ala Arg Ala Glu Gln Thr Thr Val Trp Leu 725 730 735 Cys Gly Thr Gly Thr Val Lys Thr Pro Val Leu Thr Ala Asp Glu Gly 740 745 750 Ile Leu Lys Thr Ala Ile Lys Leu Gln Ala Gln Val Gln Glu Leu Glu 755 760 765 Glu Asn Asp Glu Trp Pro Leu Glu Thr Phe Glu Lys Tyr Leu Leu Ser 770 775 780 Leu Ala Val Arg Arg Thr Pro Ile Asp Arg Val Ile Leu Phe Gly Gln 785 790 795 800 Arg Met Asp Thr Glu Leu Leu Asn Val Ala Lys Gln Ile Ile Trp Gln 805 810 815 His Val Asn Ser Lys Cys Leu Phe Val Ser Val Leu Leu Arg Lys Met 820 825 830 Gln Tyr Met Ser Pro Asn Leu Asn Pro Asn Asp Val Thr Leu Ser Gly 835 840 845 Cys Thr Asp Gly Ile Leu Lys Phe Ile Ala Glu His Gly Ala Ser Arg 850 855 860 Leu Leu Glu His Val Gly Gln Leu Asp Lys Ile Phe Lys Ile Pro Pro 865 870 875 880 Pro Pro Gly Lys Thr Lys Val Ser Pro Leu Arg Pro Leu Glu Glu Asn 885 890 895 Asn Pro Gly Pro Phe Val Pro Ile Ser Gln His Gly Trp Arg Asn Ile 900 905 910 Arg Leu Phe Ile Ser Ser Thr Phe Arg Asp Met His Gly Glu Arg Asp 915 920 925 Leu Leu Met Arg Ser Val Leu Pro Ala Leu Gln Ala Arg Ala Phe Pro 930 935 940 His Arg Ile Ser Leu His Ala Ile Asp Leu Arg Trp Gly Ile Thr Glu 945 950 955 960 Glu Glu Thr Arg Arg Asn Arg Gln Leu Glu Val Cys Leu Gly Glu Val 965 970 975 Glu Asn Ser Gln Leu Phe Val Gly Ile Leu Gly Ser Arg Tyr Gly Tyr 980 985 990 Thr Pro Pro Ser Tyr Asp Leu Pro Asp His Pro His Phe His Trp Thr 995 1000 1005 Gln Arg Tyr Pro Ser Gly Arg Ser Val Thr Glu Met Glu Val Met Gln 1010 1015 1020 Phe Leu Asn Arg Gly Gln Arg Ser Glu Pro Ser Asp Gln Ala Leu Ile 1025 1030 1035 1040 Tyr Phe Arg Asp Pro Gly Phe Leu Ser Ser Val Pro Asp Val Trp Lys 1045 1050 1055 Pro Asp Phe Ile Ser Glu Ser Glu Glu Ala Ala His Arg Val Ser Glu 1060 1065 1070 Leu Lys Arg Phe Leu Gln Glu Gln Lys Glu Val Thr Cys Arg Arg Tyr 1075 1080 1085 Ser Cys Glu Trp Gly Gly Val Ala Ala Gly Arg Pro Tyr Thr Gly Gly 1090 1095 1100 Leu Glu Glu Phe Gly Gln Leu Val Leu Gln Asp Val Trp Ser Val Ile 1105 1110 1115 1120 Gln Lys Arg Tyr Leu Gln Pro Gly Ala Gln Leu Glu Gln Pro Gly Ser 1125 1130 1135 Ile Ser Glu Glu Asp Leu Ile Gln Ala Ser Phe Gln Gln Leu Lys Ser 1140 1145 1150 Pro Pro Ser Pro Ala Arg Pro Arg Leu Leu Gln Asp Thr Val Gln Gln 1155 1160 1165 Leu Met Leu Pro His Gly Arg Leu Ser Leu Val Ile Gly Gln Ala Gly 1170 1175 1180 Gln Gly Lys Thr Ala Phe Leu Ala Ser Leu Val Ser Ala Leu Lys Val 1185 1190 1195 1200 Pro Asp Gln Pro Asn Val Ala Pro Phe Val Phe Phe His Phe Ser Ala 1205 1210 1215 Ala Arg Pro Asp Gln Cys Leu Ala Phe Asn Leu Leu Arg Arg Leu Cys 1220 1225 1230 Thr His Leu His Gln Lys Leu Gly Glu Pro Ser Ala Leu Pro Ser Thr 1235 1240 1245 Tyr Arg Gly Leu Val Trp Glu Leu Gln Gln Lys Leu Leu Leu Lys Ser 1250 1255 1260 Ala Gln Trp Leu Gln Pro Gly Gln Thr Leu Val Leu Ile Ile Asp Gly 1265 1270 1275 1280 Ala Asp Lys Leu Val Asp His Asn Gly Gln Leu Ile Ser Asp Trp Ile 1285 1290 1295 Pro Lys Ser Leu Pro Arg Arg Val His Leu Val Leu Ser Val Ser Ser 1300 1305 1310 Asp Ser Gly Leu Gly Glu Thr Leu Gln Gln Ser Gln Ser Ala Tyr Val 1315 1320 1325 Val Ala Leu Gly Ser Leu Val Pro Ser Ser Arg Ala Gln Leu Val Arg 1330 1335 1340 Glu Glu Leu Ala Leu Tyr Gly Lys Arg Leu Glu Glu Ser Pro Phe Asn 1345 1350 1355 1360 Asn Gln Met Arg Leu Leu Leu Ala Lys Gln Gly Ser Ser Leu Pro Leu 1365 1370 1375 Tyr Leu His Leu Val Thr Asp Tyr Leu Arg Leu Phe Thr Leu Tyr Glu 1380 1385 1390 Gln Val Ser Glu Arg Leu Arg Thr Leu Pro Ala Thr Leu Pro Leu Leu 1395 1400 1405 Leu Gln His Ile Leu Ser Thr Leu Glu Gln Glu His Gly His Asn Val 1410 1415 1420 Leu Pro Gln Ala Leu Thr Ala Leu Glu Val Thr His Ser Gly Leu Thr 1425 1430 1435 1440 Val Asp Gln Leu His Ala Val Leu Ser Thr Trp Leu Thr Leu Pro Lys 1445 1450 1455 Glu Thr Lys Ser Trp Glu Glu Ala Val Ala Ala Ser His Ser Gly Asn 1460 1465 1470 Leu Tyr Pro Leu Ala Pro Phe Ala Tyr Leu Val Gln Ser Leu Arg Ser 1475 1480 1485 Leu Leu Gly Glu Gly Pro Val Glu Arg Pro Gly Ala Arg Leu Cys Leu 1490 1495 1500 Ser Asp Gly Pro Leu Arg Thr Ala Val Lys Arg Arg Tyr Gly Lys Arg 1505 1510 1515 1520 Leu Gly Leu Glu Lys Thr Ala His Val Leu Ile Ala Ala His Leu Trp 1525 1530 1535 Lys Met Cys Asp Pro Asp Ala Ser Gly Thr Phe Arg Ser Cys Pro Pro 1540 1545 1550 Glu Ala Leu Lys Asp Leu Pro Tyr His Leu Leu Gln Ser Gly Asn His 1555 1560 1565 Gly Leu Leu Ala Lys Phe Leu Thr Asn Leu His Val Val Ala Ala Tyr 1570 1575 1580 Leu Glu Val Gly Leu Val Pro Asp Leu Leu Glu Ala Tyr Glu Leu Tyr 1585 1590 1595 1600 Ala Ser Ser Lys Pro Glu Val Asn Gln Lys Leu Pro Glu Ala Asp Val 1605 1610 1615 Ala Val Phe His Asn Phe Leu Lys Gln Gln Ala Ser Leu Leu Thr Gln 1620 1625 1630 Tyr Pro Leu Leu Leu Leu Gln Gln Ala Ala Ser Gln Pro Glu Glu Ser 1635 1640 1645 Pro Val Cys Cys Gln Ala Pro Leu Leu Thr Gln Arg Trp His Asn Gln 1650 1655 1660 Cys Ile Leu Lys Trp Ile Asn Lys Pro Gln Thr Leu Lys Gly Gln Gln 1665 1670 1675 1680 Ser Leu Ser Leu Pro Ile Ser Ser Ser Pro Thr Ala Val Ala Phe Ser 1685 1690 1695 Pro Asn Gly Gln Arg Ala Ala Val Gly Thr Ala Gly Gly Thr Ile Tyr 1700 1705 1710 Leu Leu Asn Leu Arg Thr Trp Gln Glu Glu Lys Ala Leu Val Ser Gly 1715 1720 1725 Cys Asp Gly Ile Ser Ser Phe Ala Phe Leu Ser Asp Thr Ala Leu Phe 1730 1735 1740 Leu Thr Thr Phe Asp Gly Leu Leu Glu Leu Trp Asp Leu Gln His Gly 1745 1750 1755 1760 Cys Trp Val Phe Gln Thr Lys Ala His Gln Tyr Gln Ile Thr Gly Cys 1765 1770 1775 Cys Leu Ser Pro Asp Arg Arg Leu Leu Ala Thr Val Cys Leu Gly Gly 1780 1785 1790 Tyr Val Lys Leu Trp Asp Thr Val Gln Gly Gln Leu Ala Phe Gln Tyr 1795 1800 1805 Thr His Pro Lys Ser Leu Asn Cys Ile Thr Phe His Pro Glu Gly Gln 1810 1815 1820 Val Val Ala Thr Gly Asn Trp Ser Gly Ile Val Thr Phe Phe Gln Ala 1825 1830 1835 1840 Asp Gly Leu Lys Val Thr Lys Glu Leu Gly Gly Pro Gly Pro Ser Val 1845 1850 1855 Arg Thr Leu Ala Phe Ser Ala Pro Gly Lys Val Val Ala Leu Gly Arg 1860 1865 1870 Ile Asp Gly Thr Val Glu Leu Trp Ala Trp Gln Glu Gly Thr Arg Leu 1875 1880 1885 Ala Ala Phe Pro Ala Gln Cys Gly Gly Val Ser Thr Val Leu Phe Leu 1890 1895 1900 His Ala Gly Gly Arg Phe Leu Thr Ala Gly Glu Asp Gly Lys Ala Gln 1905 1910 1915 1920 Leu Trp Ser Gly Phe Leu Gly Arg Pro Arg Gly Cys Leu Gly Ser Leu 1925 1930 1935 Tyr Leu Ser Pro Ala Leu Ser Val Ala Leu Asn Pro Asp Gly Asp Gln 1940 1945 1950 Val Ala Val Gly Tyr Arg Gly Asp Gly Ile Lys Ile Tyr Arg Ile Ser 1955 1960 1965 Ser Gly Pro Gln Glu Ala Gln Cys Gln Glu Leu Asn Val Ala Val Ser 1970 1975 1980 Ala Leu Val Trp Leu Ser Pro Ser Val Leu Val Ser Gly Ala Glu Asp 1985 1990 1995 2000 Gly Ser Leu His Gly Trp Met Leu Arg Arg Asn Ser Leu Gln Ser Leu 2005 2010 2015 Trp Leu Ser Ser Val Cys Gln Lys Pro Val Leu Gly Leu Ala Ala Ser 2020 2025 2030 Gln Glu Phe Leu Ala Ser Ala Ser Glu Asp Phe Thr Val Arg Leu Trp 2035 2040 2045 Pro Arg Gln Leu Leu Thr Gln Pro His Ala Val Glu Glu Leu Pro Cys 2050 2055 2060 Ala Ala Glu Leu Arg Gly His Glu Gly Pro Val Cys Cys Cys Ser Phe 2065 2070 2075 2080 Ser Pro Asp Gly Arg Ile Leu Ala Thr Ala Gly Arg Asp Arg Asn Leu 2085 2090 2095 Leu Cys Trp Asp Val Lys Val Ala Gln Ala Pro Leu Leu Ile His Thr 2100 2105 2110 Phe Ser Ser Cys His Arg Asp Trp Ile Thr Gly Cys Thr Trp Thr Lys 2115 2120 2125 Asp Asn Ile Leu Ile Ser Cys Ser Ser Asp Gly Ser Val Gly Leu Trp 2130 2135 2140 Asn Pro Glu Ala Gly Gln Gln Leu Gly Gln Phe Pro Gly His Gln Ser 2145 2150 2155 2160 Ala Val Ser Ala Val Val Ala Val Glu Glu His Ile Val Ser Val Ser 2165 2170 2175 Arg Asp Gly Thr Leu Lys Val Trp Asp Arg Gln Gly Val Glu Leu Thr 2180 2185 2190 Ser Ile Pro Ala His Ser Gly Pro Ile Ser Gln Cys Ala Ala Ala Leu 2195 2200 2205 Glu Pro Arg Pro Ala Gly Gln Pro Gly Ser Glu Leu Met Val Val Thr 2210 2215 2220 Val Gly Leu Asp Gly Ala Thr Lys Leu Trp His Pro Leu Leu Val Cys 2225 2230 2235 2240 Gln Ile His Thr Leu Gln Gly His Ser Gly Pro Val Thr Ala Ala Ala 2245 2250 2255 Ala Ser Glu Ala Ser Gly Leu Leu Leu Thr Ser Asp Asn Ser Ser Val 2260 2265 2270 Arg Leu Trp Gln Ile Pro Lys Glu Ala Asp Asp Thr Cys Lys Pro Arg 2275 2280 2285 Ser Ser Ala Val Ile Thr Ala Val Ala Trp Ala Pro Asp Gly Ser Leu 2290 2295 2300 Val Val Ser Gly Asn Glu Ala Gly Glu Leu Thr Leu Trp Gln Lys Ala 2305 2310 2315 2320 Gln Ala Val Ala Thr Ala Arg Ala Pro Gly Arg Val Ser Asp Leu Ile 2325 2330 2335 Trp Cys Ser Ala Asn Ala Phe Phe Val Leu Ser Ala Asn Glu Asn Val 2340 2345 2350 Ser Glu Trp Gln Val Glu Leu Arg Lys Gly Ser Thr Cys Thr Asn Phe 2355 2360 2365 Arg Leu Tyr Leu Lys Arg Val Leu Gln Glu Asp Leu Gly Val Leu Thr 2370 2375 2380 Gly Met Ala Leu Ala Pro Asp Gly Gln Ser Leu Ile Leu Met Lys Glu 2385 2390 2395 2400 Asp Val Glu Leu Leu Gln Met Lys Pro Gly Ser Thr Pro Ser Ser Ile 2405 2410 2415 Cys Arg Arg Tyr Ala Val His Ser Ser Ile Leu Cys Thr Ser Lys Asp 2420 2425 2430 Tyr Gly Leu Phe Tyr Leu Gln Gln Gly Asn Ser Gly Ser Leu Ser Ile 2435 2440 2445 Leu Glu Gln Glu Glu Ser Gly Lys Phe Glu Lys Thr Leu Asp Phe Asn 2450 2455 2460 Leu Asn Leu Asn Asn Pro Asn Gly Ser Pro Val Ser Ile Thr Gln Ala 2465 2470 2475 2480 Glu Pro Glu Ser Gly Ser Ser Leu Leu Cys Ala Thr Ser Asp Gly Met 2485 2490 2495 Leu Trp Asn Leu Ser Glu Cys Thr Pro Glu Gly Glu Trp Val Val Asp 2500 2505 2510 Asn Ile Trp Gln Lys Lys Ser Arg Asn Pro Lys Ser Arg Thr Pro Gly 2515 2520 2525 Thr Asp Ser Ser Pro Gly Leu Phe Cys Met Asp Ser Trp Val Glu Pro 2530 2535 2540 Thr His Leu Lys Ala Arg Gln Cys Lys Lys Ile His Leu Gly Ser Val 2545 2550 2555 2560 Thr Ala Leu His Val Leu Pro Gly Leu Leu Val Thr Ala Ser Glu Asp 2565 2570 2575 Arg Asp Val Lys Leu Trp Glu Arg Pro Ser Met Gln Leu Leu Gly Leu 2580 2585 2590 Phe Arg Cys Glu Gly Pro Val Ser Cys Leu Glu Pro Trp Met Glu Pro 2595 2600 2605 Ser Ser Pro Leu Gln Leu Ala Val Gly Asp Ala Gln Gly Asn Leu Tyr 2610 2615 2620 Phe Leu Ser Trp Glu 2625 55 2629 PRT Rattus Norvegicus 55 Met Glu Lys Leu Cys Gly Tyr Val Pro Val His Pro Asp Ile Leu Ser 1 5 10 15 Leu Lys Asn Arg Cys Leu Thr Met Leu Ser Asp Ile Gln Pro Leu Glu 20 25 30 Lys Ile His Gly Gln Arg Ser Val Asn Pro Asp Ile Leu Ser Leu Glu 35 40 45 Asn Arg Cys Leu Thr Leu Leu Pro Asp Leu Gln Pro Met Glu Lys Ile 50 55 60 His Gly Gln Arg Ser Val His Pro Asp Ile Leu Ser Ser Glu Asn Arg 65 70 75 80 Cys Leu Thr Leu Leu Pro Asp Leu Gln Ser Leu Glu Lys Leu Cys Gly 85 90 95 His Met Ser Ser His Pro Asp Val Leu Ser Leu Glu Asn Arg Cys Leu 100 105 110 Ala Thr Leu Pro Thr Val Lys Arg Thr Val Ser Ser Gly Pro Leu Leu 115 120 125 Gln Cys Leu His Arg Ser His Thr Ala Gln Ala Asp Leu Arg Asp Pro 130 135 140 Asn Phe Arg Asn Cys Leu Phe Pro Glu Pro Pro Thr Ile Glu Ala Pro 145 150 155 160 Cys Phe Leu Lys Glu Leu Asp Leu Pro Thr Gly Pro Arg Ala Leu Lys 165 170 175 Ser Met Ser Ala Thr Ala Arg Val Gln Glu Val Ala Leu Gly Gln Arg 180 185 190 Cys Val Ser Glu Gly Lys Glu Leu Gln Glu Glu Lys Glu Ser Ala Glu 195 200 205 Val Pro Met Pro Leu Tyr Ser Leu Ser Leu Gly Gly Glu Glu Glu Glu 210 215 220 Val Val Gly Ala Pro Val Leu Lys Leu Thr Ser Gly Asp Ser Asp Ser 225 230 235 240 His Pro Glu Thr Thr Asp Gln Ile Leu Gln Glu Lys Lys Met Ala Leu 245 250 255 Leu Thr Leu Leu Cys Ser Ala Met Ala Ser Ser Val Asn Val Lys Asp 260 265 270 Ala Ser Asp Pro Thr Arg Ala Ser Ile His Glu Val Cys Ser Ala Leu 275 280 285 Ala Pro Leu Glu Pro Glu Phe Ile Leu Lys Ala Ser Leu Tyr Ala Arg 290 295 300 Gln Gln Leu Asn Leu Arg Asp Ile Ala Asn Ile Val Leu Ala Val Ala 305 310 315 320 Ala Leu Leu Pro Ala Cys Arg Pro His Val Arg Arg Tyr Tyr Ser Ala 325 330 335 Ile Val His Leu Pro Ser Asp Trp Ile Gln Val Ala Glu Phe Tyr Gln 340 345 350 Ser Leu Ala Glu Gly Asp Glu Lys Lys Leu Val Pro Leu Pro Ala Cys 355 360 365 Leu Arg Ala Ala Met Thr Asp Lys Phe Ala Gln Phe Asp Glu Tyr Gln 370 375 380 Leu Ala Lys Tyr Asn Pro Arg Lys His Arg Ser Lys Thr Arg Ser Arg 385 390 395 400 Gln Pro Pro Arg Pro Gln Arg Thr Lys Pro Pro Phe Ser Glu Ser Gly 405 410 415 Lys Cys Phe Pro Lys Ser Val Trp Pro Leu Lys Asn Glu Gln Ile Ser 420 425 430 Phe Glu Ala Ala Tyr Asn Ala Val Ser Glu Lys Lys Arg Leu Pro Arg 435 440 445 Phe Thr Leu Lys Lys Leu Val Glu Gln Leu His Ile His Glu Pro Ala 450 455 460 Gln His Val Gln Ala Leu Leu Gly Tyr Arg Tyr Pro Ser Thr Leu Glu 465 470 475 480 Leu Phe Ser Arg Ser His Leu Pro Gly Pro Trp Asp Ser Ser Arg Ala 485 490 495 Gly Gln Arg Met Lys Leu Gln Arg Pro Glu Thr Trp Glu Arg Glu Leu 500 505 510 Ser Leu Arg Gly Asn Arg Ala Ser Val Trp Glu Glu Leu Ile Asp Asn 515 520 525 Gly Lys Leu Pro Phe Met Ala Met Leu Arg Asn Leu Cys Asn Leu Leu 530 535 540 Arg Thr Gly Ile Ser Ala His His His Glu Leu Val Leu Gln Arg Leu 545 550 555 560 Gln His Glu Lys Ser Val Ile His Ser Arg Gln Phe Pro Phe Arg Phe 565 570 575 Leu Asn Ala His Asp Ser Leu Asp Arg Leu Glu Ala Gln Leu Arg Ser 580 585 590 Lys Ala Ser Pro Phe Pro Ser Asn Thr Thr Leu Met Lys Arg Ile Met 595 600 605 Ile Arg Asn Ser Lys Lys Ile Lys Arg Pro Ala Asn Pro Arg Tyr Leu 610 615 620 Cys Thr Leu Thr Gln Arg Gln Leu Arg Ala Ala Met Ala Ile Pro Val 625 630 635 640 Met Tyr Glu His Leu Lys Arg Glu Lys Leu Arg Leu His Lys Ala Arg 645 650 655 Gln Trp Thr Cys Asp Leu Glu Leu Leu Glu Arg Tyr Arg Gln Ala Leu 660 665 670 Glu Thr Ala Val Asn Ile Ser Val Lys His Asn Leu Pro Pro Leu Pro 675 680 685 Gly Arg Thr Leu Leu Val Tyr Leu Thr Asp Ala Asn Ala Asn Arg Leu 690 695 700 Cys Pro Lys Ser His Leu Gln Gly Pro Pro Leu Asn Tyr Val Leu Leu 705 710 715 720 Leu Ile Gly Met Met Met Ala Arg Ala Glu Gln Thr Thr Val Trp Leu 725 730 735 Cys Gly Thr Gly Thr Val Lys Thr Pro Val Leu Thr Ala Asp Glu Gly 740 745 750 Ile Leu Lys Thr Ala Ile Lys Leu Gln Ala Gln Val Gln Glu Leu Glu 755 760 765 Glu Asn Asp Glu Trp Pro Leu Glu Thr Phe Glu Lys Tyr Leu Leu Ser 770 775 780 Leu Ala Val Arg Arg Thr Pro Ile Asp Arg Val Ile Leu Phe Gly Gln 785 790 795 800 Arg Met Asp Thr Glu Leu Leu Asn Val Ala Lys Gln Ile Ile Trp Gln 805 810 815 His Val Asn Ser Lys Cys Leu Phe Val Ser Val Leu Leu Arg Lys Met 820 825 830 Gln Tyr Met Ser Pro Asn Leu Asn Pro Asn Asp Val Thr Leu Ser Gly 835 840 845 Cys Thr Asp Gly Ile Leu Lys Phe Ile Ala Glu His Gly Ala Ser Arg 850 855 860 Leu Leu Glu His Val Gly Gln Leu Asp Lys Ile Phe Lys Ile Pro Pro 865 870 875 880 Pro Pro Gly Lys Thr Lys Val Ser Pro Leu Arg Pro Leu Glu Glu Asn 885 890 895 Asn Pro Gly Pro Phe Val Pro Ile Ser Gln His Gly Trp Arg Asn Ile 900 905 910 Arg Leu Phe Ile Ser Ser Thr Phe Arg Asp Met His Gly Glu Arg Asp 915 920 925 Leu Leu Met Arg Ser Val Leu Pro Ala Leu Gln Ala Arg Ala Phe Pro 930 935 940 His Arg Ile Ser Leu His Ala Ile Asp Leu Arg Trp Gly Ile Thr Glu 945 950 955 960 Glu Glu Thr Arg Arg Asn Arg Gln Leu Glu Val Cys Leu Gly Glu Val 965 970 975 Glu Asn Ser Gln Leu Phe Val Gly Ile Leu Gly Ser Arg Tyr Gly Tyr 980 985 990 Thr Pro Pro Ser Tyr Asp Leu Pro Asp His Pro His Phe His Trp Thr 995 1000 1005 Gln Arg Tyr Pro Ser Gly Arg Ser Val Thr Glu Met Glu Val Met Gln 1010 1015 1020 Phe Leu Asn Arg Gly Gln Arg Ser Glu Pro Ser Asp Gln Ala Leu Ile 1025 1030 1035 1040 Tyr Phe Arg Asp Pro Gly Phe Leu Ser Ser Val Pro Asp Val Trp Lys 1045 1050 1055 Pro Asp Phe Ile Ser Glu Ser Glu Glu Ala Ala His Arg Val Ser Glu 1060 1065 1070 Leu Lys Arg Phe Leu Gln Glu Gln Lys Glu Val Thr Cys Arg Arg Tyr 1075 1080 1085 Ser Cys Glu Trp Gly Gly Val Ala Ala Gly Arg Pro Tyr Thr Gly Gly 1090 1095 1100 Leu Glu Glu Phe Gly Gln Leu Val Leu Gln Asp Val Trp Ser Val Ile 1105 1110 1115 1120 Gln Lys Arg Tyr Leu Gln Pro Gly Ala Gln Leu Glu Gln Pro Gly Ser 1125 1130 1135 Ile Ser Glu Glu Asp Leu Ile Gln Ala Ser Phe Gln Gln Leu Lys Ser 1140 1145 1150 Pro Pro Ser Pro Ala Arg Pro Arg Leu Leu Gln Asp Thr Val Gln Gln 1155 1160 1165 Leu Met Leu Pro His Gly Arg Leu Ser Leu Val Ile Gly Gln Ala Gly 1170 1175 1180 Gln Gly Lys Thr Ala Phe Leu Ala Ser Leu Val Ser Ala Leu Lys Val 1185 1190 1195 1200 Pro Asp Gln Pro Asn Val Ala Pro Phe Val Phe Phe His Phe Ser Ala 1205 1210 1215 Ala Arg Pro Asp Gln Cys Leu Ala Phe Asn Leu Leu Arg Arg Leu Cys 1220 1225 1230 Thr His Leu His Gln Lys Leu Gly Glu Pro Ser Ala Leu Pro Ser Thr 1235 1240 1245 Tyr Arg Gly Leu Val Trp Glu Leu Gln Gln Lys Leu Leu Leu Lys Ser 1250 1255 1260 Ala Gln Trp Leu Gln Pro Gly Gln Thr Leu Val Leu Ile Ile Asp Gly 1265 1270 1275 1280 Ala Asp Lys Leu Val Asp His Asn Gly Gln Leu Ile Ser Asp Trp Ile 1285 1290 1295 Pro Lys Ser Leu Pro Arg Arg Val His Leu Val Leu Ser Val Ser Ser 1300 1305 1310 Asp Ser Gly Leu Gly Glu Thr Leu Gln Gln Ser Gln Ser Ala Tyr Val 1315 1320 1325 Val Ala Leu Gly Ser Leu Val Pro Ser Ser Arg Ala Gln Leu Val Arg 1330 1335 1340 Glu Glu Leu Ala Leu Tyr Gly Lys Arg Leu Glu Glu Ser Pro Phe Asn 1345 1350 1355 1360 Asn Gln Met Arg Leu Leu Leu Ala Lys Gln Gly Ser Ser Leu Pro Leu 1365 1370 1375 Tyr Leu His Leu Val Thr Asp Tyr Leu Arg Leu Phe Thr Leu Tyr Glu 1380 1385 1390 Gln Val Ser Glu Arg Leu Arg Thr Leu Pro Ala Thr Leu Pro Leu Leu 1395 1400 1405 Leu Gln His Ile Leu Ser Thr Leu Glu Gln Glu His Gly His Asn Val 1410 1415 1420 Leu Pro Gln Ala Leu Thr Ala Leu Glu Val Thr His Ser Gly Leu Thr 1425 1430 1435 1440 Val Asp Gln Leu His Ala Val Leu Ser Thr Trp Leu Thr Leu Pro Lys 1445 1450 1455 Glu Thr Lys Ser Trp Glu Glu Ala Val Ala Ala Ser His Ser Gly Asn 1460 1465 1470 Leu Tyr Pro Leu Ala Pro Phe Ala Tyr Leu Val Gln Ser Leu Arg Ser 1475 1480 1485 Leu Leu Gly Glu Gly Pro Val Glu Arg Pro Gly Ala Arg Leu Cys Leu 1490 1495 1500 Ser Asp Gly Pro Leu Arg Thr Ala Val Lys Arg Arg Tyr Gly Lys Arg 1505 1510 1515 1520 Leu Gly Leu Glu Lys Thr Ala His Val Leu Ile Ala Ala His Leu Trp 1525 1530 1535 Lys Met Cys Asp Pro Asp Ala Ser Gly Thr Phe Arg Ser Cys Pro Pro 1540 1545 1550 Glu Ala Leu Lys Asp Leu Pro Tyr His Leu Leu Gln Ser Gly Asn His 1555 1560 1565 Gly Leu Leu Ala Lys Phe Leu Thr Asn Leu His Val Val Ala Ala Tyr 1570 1575 1580 Leu Glu Val Gly Leu Val Pro Asp Leu Leu Glu Ala Tyr Glu Leu Tyr 1585 1590 1595 1600 Ala Ser Ser Lys Pro Glu Val Asn Gln Lys Leu Pro Glu Ala Asp Val 1605 1610 1615 Ala Val Phe His Asn Phe Leu Lys Gln Gln Ala Ser Leu Leu Thr Gln 1620 1625 1630 Tyr Pro Leu Leu Leu Leu Gln Gln Ala Ala Ser Gln Pro Glu Glu Ser 1635 1640 1645 Pro Val Cys Cys Gln Ala Pro Leu Leu Thr Gln Arg Trp His Asn Gln 1650 1655 1660 Cys Ile Leu Lys Trp Ile Asn Lys Pro Gln Thr Leu Lys Gly Gln Gln 1665 1670 1675 1680 Ser Leu Ser Leu Pro Ile Ser Ser Ser Pro Thr Ala Val Ala Phe Ser 1685 1690 1695 Pro Asn Gly Gln Arg Ala Ala Val Gly Thr Ala Gly Gly Thr Ile Tyr 1700 1705 1710 Leu Leu Asn Leu Arg Thr Trp Gln Glu Glu Lys Ala Leu Val Ser Gly 1715 1720 1725 Cys Asp Gly Ile Ser Ser Phe Ala Phe Leu Ser Asp Thr Ala Leu Phe 1730 1735 1740 Leu Thr Thr Phe Asp Gly Leu Leu Glu Leu Trp Asp Leu Gln His Gly 1745 1750 1755 1760 Cys Trp Val Phe Gln Thr Lys Ala His Gln Tyr Gln Ile Thr Gly Cys 1765 1770 1775 Cys Leu Ser Pro Asp Arg Arg Leu Leu Ala Thr Val Cys Leu Gly Gly 1780 1785 1790 Tyr Val Lys Leu Trp Asp Thr Val Gln Gly Gln Leu Ala Phe Gln Tyr 1795 1800 1805 Thr His Pro Lys Ser Leu Asn Cys Ile Thr Phe His Pro Glu Gly Gln 1810 1815 1820 Val Val Ala Thr Gly Asn Trp Ser Gly Ile Val Thr Phe Phe Gln Ala 1825 1830 1835 1840 Asp Gly Leu Lys Val Thr Lys Glu Leu Gly Gly Pro Gly Pro Ser Val 1845 1850 1855 Arg Thr Leu Ala Phe Ser Ala Pro Gly Lys Val Val Ala Leu Gly Arg 1860 1865 1870 Ile Asp Gly Thr Val Glu Leu Trp Ala Trp Gln Glu Gly Thr Arg Leu 1875 1880 1885 Ala Ala Phe Pro Ala Gln Cys Gly Gly Val Ser Thr Val Leu Phe Leu 1890 1895 1900 His Ala Gly Gly Arg Phe Leu Thr Ala Gly Glu Asp Gly Lys Ala Gln 1905 1910 1915 1920 Leu Trp Ser Gly Phe Leu Gly Arg Pro Arg Gly Cys Leu Gly Ser Leu 1925 1930 1935 Tyr Leu Ser Pro Ala Leu Ser Val Ala Leu Asn Pro Asp Gly Asp Gln 1940 1945 1950 Val Ala Val Gly Tyr Arg Gly Asp Gly Ile Lys Ile Tyr Arg Ile Ser 1955 1960 1965 Ser Gly Pro Gln Glu Ala Gln Cys Gln Glu Leu Asn Val Ala Val Ser 1970 1975 1980 Ala Leu Val Trp Leu Ser Pro Ser Val Leu Val Ser Gly Ala Glu Asp 1985 1990 1995 2000 Gly Ser Leu His Gly Trp Met Leu Arg Arg Asn Ser Leu Gln Ser Leu 2005 2010 2015 Trp Leu Ser Ser Val Cys Gln Lys Pro Val Leu Gly Leu Ala Ala Ser 2020 2025 2030 Gln Glu Phe Leu Ala Ser Ala Ser Glu Asp Phe Thr Val Arg Leu Trp 2035 2040 2045 Pro Arg Gln Leu Leu Thr Gln Pro His Ala Val Glu Glu Leu Pro Cys 2050 2055 2060 Ala Ala Glu Leu Arg Gly His Glu Gly Pro Val Cys Cys Cys Ser Phe 2065 2070 2075 2080 Ser Pro Asp Gly Arg Ile Leu Ala Thr Ala Gly Arg Asp Arg Asn Leu 2085 2090 2095 Leu Cys Trp Asp Val Lys Val Ala Gln Ala Pro Leu Leu Ile His Thr 2100 2105 2110 Phe Ser Ser Cys His Arg Asp Trp Ile Thr Gly Cys Thr Trp Thr Lys 2115 2120 2125 Asp Asn Ile Leu Ile Ser Cys Ser Ser Asp Gly Ser Val Gly Leu Trp 2130 2135 2140 Asn Pro Glu Ala Gly Gln Gln Leu Gly Gln Phe Pro Gly His Gln Ser 2145 2150 2155 2160 Ala Val Ser Ala Val Val Ala Val Glu Glu His Ile Val Ser Val Ser 2165 2170 2175 Arg Asp Gly Thr Leu Lys Val Trp Asp Arg Gln Gly Val Glu Leu Thr 2180 2185 2190 Ser Ile Pro Ala His Ser Gly Pro Ile Ser Gln Cys Ala Ala Ala Leu 2195 2200 2205 Glu Pro Arg Pro Ala Gly Gln Pro Gly Ser Glu Leu Met Val Val Thr 2210 2215 2220 Val Gly Leu Asp Gly Ala Thr Lys Leu Trp His Pro Leu Leu Val Cys 2225 2230 2235 2240 Gln Ile His Thr Leu Gln Gly His Ser Gly Pro Val Thr Ala Ala Ala 2245 2250 2255 Ala Ser Glu Ala Ser Gly Leu Leu Leu Thr Ser Asp Asn Ser Ser Val 2260 2265 2270 Arg Leu Trp Gln Ile Pro Lys Glu Ala Asp Asp Thr Cys Lys Pro Arg 2275 2280 2285 Ser Ser Ala Val Ile Thr Ala Val Ala Trp Ala Pro Asp Gly Ser Leu 2290 2295 2300 Val Val Ser Gly Asn Glu Ala Gly Glu Leu Thr Leu Trp Gln Lys Ala 2305 2310 2315 2320 Gln Ala Val Ala Thr Ala Arg Ala Pro Gly Arg Val Ser Asp Leu Ile 2325 2330 2335 Trp Cys Ser Ala Asn Ala Phe Phe Val Leu Ser Ala Asn Glu Asn Val 2340 2345 2350 Ser Glu Trp Gln Val Glu Leu Arg Lys Gly Ser Thr Cys Thr Asn Phe 2355 2360 2365 Arg Leu Tyr Leu Lys Arg Val Leu Gln Glu Asp Leu Gly Val Leu Thr 2370 2375 2380 Gly Met Ala Leu Ala Pro Asp Gly Gln Ser Leu Ile Leu Met Lys Glu 2385 2390 2395 2400 Asp Val Glu Leu Leu Gln Met Lys Pro Gly Ser Thr Pro Ser Ser Ile 2405 2410 2415 Cys Arg Arg Tyr Ala Val His Ser Ser Ile Leu Cys Thr Ser Lys Asp 2420 2425 2430 Tyr Gly Leu Phe Tyr Leu Gln Gln Gly Asn Ser Gly Ser Leu Ser Ile 2435 2440 2445 Leu Glu Gln Glu Glu Ser Gly Lys Phe Glu Lys Thr Leu Asp Phe Asn 2450 2455 2460 Leu Asn Leu Asn Asn Pro Asn Gly Ser Pro Val Ser Ile Thr Gln Ala 2465 2470 2475 2480 Glu Pro Glu Ser Gly Ser Ser Leu Leu Cys Ala Thr Ser Asp Gly Met 2485 2490 2495 Leu Trp Asn Leu Ser Glu Cys Thr Pro Glu Gly Glu Trp Val Val Asp 2500 2505 2510 Asn Ile Trp Gln Lys Lys Ser Arg Asn Pro Lys Ser Arg Thr Pro Gly 2515 2520 2525 Thr Asp Ser Ser Pro Gly Leu Phe Cys Met Asp Ser Trp Val Glu Pro 2530 2535 2540 Thr His Leu Lys Ala Arg Gln Cys Lys Lys Ile His Leu Gly Ser Val 2545 2550 2555 2560 Thr Ala Leu His Val Leu Pro Gly Leu Leu Val Thr Ala Ser Glu Asp 2565 2570 2575 Arg Asp Val Lys Leu Trp Glu Arg Pro Ser Met Gln Leu Leu Gly Leu 2580 2585 2590 Phe Arg Cys Glu Gly Pro Val Ser Cys Leu Glu Pro Trp Met Glu Pro 2595 2600 2605 Ser Ser Pro Leu Gln Leu Ala Val Gly Asp Ala Gln Gly Asn Leu Tyr 2610 2615 2620 Phe Leu Ser Trp Glu 2625 56 4425 DNA Cryptosporidium parvum 56 atgagtgtgt tgaagggtgc tttagcagtg ttgatgaagc aagaggttct gacgctggga 60 gagtacctgg agcagaaaag aaagattaga gtattcaaag aagaaggatt gtacaatgat 120 tcctcagggg agttaaggag gctacttgaa gagactttga tactggaaga tccttctata 180 ccaaaagctt taaaggaggg atttcttggc ttaagggaac ttacatttaa ttccttagac 240 aatagtcttg atattaacgg agacaggagg aagttgttgg gtataaatat cgaaaaattg 300 ctcgagtggt catcgatact aggtcagaac tactttagct cacctagaaa tcagtcgtct 360 atgtcttcag gaaagaggtc aagaaatacc gaatataatg agacattgtc tccttccttt 420 attacagacc aaattaagaa tataattcca gattctgaaa aggattctct gacctatcaa 480 gtacaagact ttgagctcct actccgtaag cactttccaa ggattaggat ctttcattca 540 gggagttatt caggattaag aactagcttc gacactataa agcccaagaa atactcattc 600 tgtaattcta ctttatacaa gtttcaaaac ctccatgctt ggaatactct tctttccaaa 660 ataggtccta ttgagattct cttcttattt gtttgctgta ttatctttag aatacttggt 720 gatcactctg aaattctcat tcaacaagct ggtagaatgt taactaatga cttcctggaa 780 gagcttgcta gactttatga gacagggcca aaaaagacca attcatttgt atcatcatct 840 tctttaactc caattcttac aattgaggaa aaagaagaag ttaagccttt agatgaaact 900 ccaagacaag taaacaaaag atcaggagga attattaaag attcaagact ttctaaaatc 960 tacaaaattg atattccata tcgctcaaca tttctttatt gtgatcattt ctcaaaaaga 1020 ggaggtcttc cttgtttatc tgttctaaga cttttacctc caaatttatt aggagcaaga 1080 acactactta gatttattat tcaaactgat catatattca aagatcacaa cagacaaagt 1140 ttgattggat tacttggaag ttatgaaatg acaaaattct cccgtgttaa atgtaaattg 1200 gcttctgcta tgatggaaga attccaaaat ttattgttaa atattagaaa tacgtctcca 1260 gttaactttc tagatcaaat atgtccaata gaaccaatta aagattcaga tttacaaaac 1320 ttcaataaat tacctattcc ttgttttgaa acaagttcta ctagagtagt aaatttttta 1380 agattatact tgattaaagt attaccaaag aatatacttg gtacctttaa aaactttaag 1440 acttttatta ataaaaagat cccaataatt gtgaatcttc atattagaga aacttttaag 1500 attcaacatg caatgaatgg aattgaggtt tcaaattggg taaatagaat acttgaagaa 1560 aaaagttatc aatttattaa agaatcaaaa aatttaatta attctaatca aagatctaaa 1620 aaggtaccga ataataagac aagatctagt attaaaaaaa gtttaattag ccttggaaaa 1680 acatatctag ctaggaacat ttatttttta atagtatatt tggtatttcc aatattaaga 1740 agacatttct atgcaacaga aattgagggt agtaataaag taagatattt tagacatcca 1800 gtttggataa agatcgtgag acaagctgat aaatggtatc tagaaagtat attaaaagga 1860 attcagtcaa aagactatat aatgaattta tcttcaattc aagaaatttc aaaattaatt 1920 gataagaact ctgagttttc tgaaaatatc ccaaaaataa gatgggtacc gaaatctaaa 1980 ggtttaagac cattaataaa tttatcaaaa gttggatctg gacaaattct tcaacaaaat 2040 cttgaaagag atcacacttg tgaagaaaag aagaagattg gtttttgtga ttgtaattca 2100 gtttggactg gtggagatct tccaacaagt tggattaata acaattacta ttataactac 2160 tttaatagta atggtaataa cggtattgta ggaaataatc ataattatgg tcatattaat 2220 aatataggag gaaagaattt tacatcattt tcaagccaaa attgtcagat tatgacacca 2280 ccaggaagat taggagtggg attaggattt ggtaattata atataggaat tggaataaat 2340 tcaggatcat cggtacctac tactccaatt aataaggtaa tgagacgtcc ttcaagtaat 2400 agaattatag atataagacg tccatcaaca aataatatgt tgttttatcc ttccaagatt 2460 ttaagatctt ttctattaag aatgacagga aagaatcatc ttggtgcttc tcttgtacag 2520 tttggagata tttataagat tataaagaat tggtggataa agaatagaag agacaaacaa 2580 caatttcaaa tgaatgaaga tttttatcaa aatcaaggta aaaaaatgtc ccatgtaaag 2640 atttatatta ttaaagcaga tttagtgaac tgttttgaaa atataaataa gtccaagatt 2700 tttgagtttt tggatgtaat ttctcttcca aatgaaattt cccttttgag cttatattca 2760 agggctttaa gtaaaacaac aattattccg ccttttgata atatgaatag agactcttac 2820 caagatgagc ttgggagatc atgtattatt acttcaaaag gaaaactaaa tatggtacct 2880 atttttgaga aagatcatga tgaagatcaa aaagtcgtca aatcaaaaat taagaggatt 2940 ataggaccag atttagatga acttctttgg aatcttaaaa attcttgttt atcaaacaag 3000 gatttgagta ttttgggaca aaaaaaagca gaaatcttta cttttttaaa ttcaagacgt 3060 gtgataaact ggaaattagt caaggagatc attaagatcc accttaacac aagctttttt 3120 cgacttagga catgttcaaa gtctttaaga ctaataaaat ctaaacaact tggaaattct 3180 atcaaatttg gaaaaagatt tctaagctta tttaagcaag attttggaat tccacaagga 3240 tcaagtattt cttacattct ttgttgtctc tattataatt tcttagatct aaatccagaa 3300 atccagaatc ttttgggaca ttcatttttt tcctcctctt acatttcttt atcatttctg 3360 aatccaatta aacaagaaaa agtacaatta cttgatccaa ctgaaaaaga cttattttca 3420 caacatgatc ttgagacagg attatcccgt tacccagaat ttaaagaaaa tgagataaat 3480 atttataata ctagtacaag taaaaaaaga agactagaaa tatcagaata taatattaat 3540 gctaaaagtg ttttactgca accttccgaa gagtttaata ataatatcaa caaccagaat 3600 tgccaaatcc gtgccaatct caaaagttat gatcaagaaa gtccttctca gagcaataag 3660 caagaaagtc tcttattgag atgggtagac gattttcttt ttctcacatc cgatttggag 3720 tccgccaaaa agttcttaaa gttactttat atacaaaaac tatggggatc gaacgtttcc 3780 aaggacaaaa tcaactctaa ctttccttgg attgaccaca ataatgagat catcatcctt 3840 gaggaggatg atgttttact atcttcgcct tcctcttcct gttcatcatc ttgttcctct 3900 tcatcttcct cttcatcttc ttcgtcgtct tcctcatcgt cctcttcttc cttcccacct 3960 tcgacagagt gcacaagcag tccagtggaa aagagaataa ataccaacaa aagtgaaaag 4020 gactgccaaa atgaaaaaga aatgattaac aaggctaaga tagcctcaaa acagttccat 4080 aaacaagtat catggacagg aatgaagttt tccagtgagt acagctattt gaattgcatg 4140 gtttccccat ggaagaattt ggaattcata tgtgttatgg atactgtaac cttgacaact 4200 aagcaccaat ttacaagtaa ttattcaaat tttcacaggt ttaagtcgac gacgatatct 4260 gaaaaccttc aaaagtcaaa ttatatgtgg tctgttttgg gtataaaatt aataagatac 4320 tttgatttta gaatcaagaa tggattgcta tatgattgta aaatcaattc tttatttacg 4380 gtaagtttta aatataattc aacaaataac caaccaatta attaa 4425 57 1132 PRT Homo sapiens 57 Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser 1 5 10 15 His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly 20 25 30 Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg 35 40 45 Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro 50 55 60 Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu 65 70 75 80 Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val 85 90 95 Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro 100 105 110 Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125 Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135 140 Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val 145 150 155 160 Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr 165 170 175 Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly 180 185 190 Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg 195 200 205 Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg 210 215 220 Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg 225 230 235 240 Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp 245 250 255 Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260 265 270 Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala 275 280 285 Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His 290 295 300 Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro 305 310 315 320 Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly 325 330 335 Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro 340 345 350 Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser 355 360 365 Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln 370 375 380 Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His 385 390 395 400 Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg 405 410 415 Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln 420 425 430 Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu 435 440 445 Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe 450 455 460 Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser 465 470 475 480 Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser 485 490 495 Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met 500 505 510 Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys 515 520 525 Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe 530 535 540 Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe 545 550 555 560 Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr 565 570 575 Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His 580 585 590 Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln 595 600 605 His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile 610 615 620 Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val 625 630 635 640 Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645 650 655 Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg 660 665 670 Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg 675 680 685 Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro 690 695 700 Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile 705 710 715 720 Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730 735 Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740 745 750 Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760 765 Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser 770 775 780 Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu 785 790 795 800 Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His 805 810 815 Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro 820 825 830 Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845 Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu 850 855 860 Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala 865 870 875 880 Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys 885 890 895 Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu 900 905 910 Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe 915 920 925 Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser 930 935 940 Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe 945 950 955 960 Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly 965 970 975 Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn 980 985 990 Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln 995 1000 1005 Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln 1010 1015 1020 Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala 1025 1030 1035 1040 Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu 1045 1050 1055 Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln Trp 1060 1065 1070 Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr 1075 1080 1085 Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser 1090 1095 1100 Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn 1105 1110 1115 1120 Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp 1125 1130 58 4425 DNA Cryptosporidium parvum 58 atgagtgtgt tgaagggtgc tttagcagtg ttgatgaagc aagaggttct gacgctggga 60 gagtacctgg agcagaaaag aaagattaga gtattcaaag aagaaggatt gtacaatgat 120 tcctcagggg agttaaggag gctacttgaa gagactttga tactggaaga tccttctata 180 ccaaaagctt taaaggaggg atttcttggc ttaagggaac ttacatttaa ttccttagac 240 aatagtcttg atattaacgg agacaggagg aagttgttgg gtataaatat cgaaaaattg 300 ctcgagtggt catcgatact aggtcagaac tactttagct cacctagaaa tcagtcgtct 360 atgtcttcag gaaagaggtc aagaaatacc gaatataatg agacattgtc tccttccttt 420 attacagacc aaattaagaa tataattcca gattctgaaa aggattctct gacctatcaa 480 gtacaagact ttgagctcct actccgtaag cactttccaa ggattaggat ctttcattca 540 gggagttatt caggattaag aactagcttc gacactataa agcccaagaa atactcattc 600 tgtaattcta ctttatacaa gtttcaaaac ctccatgctt ggaatactct tctttccaaa 660 ataggtccta ttgagattct cttcttattt gtttgctgta ttatctttag aatacttggt 720 gatcactctg aaattctcat tcaacaagct ggtagaatgt taactaatga cttcctggaa 780 gagcttgcta gactttatga gacagggcca aaaaagacca attcatttgt atcatcatct 840 tctttaactc caattcttac aattgaggaa aaagaagaag ttaagccttt agatgaaact 900 ccaagacaag taaacaaaag atcaggagga attattaaag attcaagact ttctaaaatc 960 tacaaaattg atattccata tcgctcaaca tttctttatt gtgatcattt ctcaaaaaga 1020 ggaggtcttc cttgtttatc tgttctaaga cttttacctc caaatttatt aggagcaaga 1080 acactactta gatttattat tcaaactgat catatattca aagatcacaa cagacaaagt 1140 ttgattggat tacttggaag ttatgaaatg acaaaattct cccgtgttaa atgtaaattg 1200 gcttctgcta tgatggaaga attccaaaat ttattgttaa atattagaaa tacgtctcca 1260 gttaactttc tagatcaaat atgtccaata gaaccaatta aagattcaga tttacaaaac 1320 ttcaataaat tacctattcc ttgttttgaa acaagttcta ctagagtagt aaatttttta 1380 agattatact tgattaaagt attaccaaag aatatacttg gtacctttaa aaactttaag 1440 acttttatta ataaaaagat cccaataatt gtgaatcttc atattagaga aacttttaag 1500 attcaacatg caatgaatgg aattgaggtt tcaaattggg taaatagaat acttgaagaa 1560 aaaagttatc aatttattaa agaatcaaaa aatttaatta attctaatca aagatctaaa 1620 aaggtaccga ataataagac aagatctagt attaaaaaaa gtttaattag ccttggaaaa 1680 acatatctag ctaggaacat ttatttttta atagtatatt tggtatttcc aatattaaga 1740 agacatttct atgcaacaga aattgagggt agtaataaag taagatattt tagacatcca 1800 gtttggataa agatcgtgag acaagctgat aaatggtatc tagaaagtat attaaaagga 1860 attcagtcaa aagactatat aatgaattta tcttcaattc aagaaatttc aaaattaatt 1920 gataagaact ctgagttttc tgaaaatatc ccaaaaataa gatgggtacc gaaatctaaa 1980 ggtttaagac cattaataaa tttatcaaaa gttggatctg gacaaattct tcaacaaaat 2040 cttgaaagag atcacacttg tgaagaaaag aagaagattg gtttttgtga ttgtaattca 2100 gtttggactg gtggagatct tccaacaagt tggattaata acaattacta ttataactac 2160 tttaatagta atggtaataa cggtattgta ggaaataatc ataattatgg tcatattaat 2220 aatataggag gaaagaattt tacatcattt tcaagccaaa attgtcagat tatgacacca 2280 ccaggaagat taggagtggg attaggattt ggtaattata atataggaat tggaataaat 2340 tcaggatcat cggtacctac tactccaatt aataaggtaa tgagacgtcc ttcaagtaat 2400 agaattatag atataagacg tccatcaaca aataatatgt tgttttatcc ttccaagatt 2460 ttaagatctt ttctattaag aatgacagga aagaatcatc ttggtgcttc tcttgtacag 2520 tttggagata tttataagat tataaagaat tggtggataa agaatagaag agacaaacaa 2580 caatttcaaa tgaatgaaga tttttatcaa aatcaaggta aaaaaatgtc ccatgtaaag 2640 atttatatta ttaaagcaga tttagtgaac tgttttgaaa atataaataa gtccaagatt 2700 tttgagtttt tggatgtaat ttctcttcca aatgaaattt cccttttgag cttatattca 2760 agggctttaa gtaaaacaac aattattccg ccttttgata atatgaatag agactcttac 2820 caagatgagc ttgggagatc atgtattatt acttcaaaag gaaaactaaa tatggtacct 2880 atttttgaga aagatcatga tgaagatcaa aaagtcgtca aatcaaaaat taagaggatt 2940 ataggaccag atttagatga acttctttgg aatcttaaaa attcttgttt atcaaacaag 3000 gatttgagta ttttgggaca aaaaaaagca gaaatcttta cttttttaaa ttcaagacgt 3060 gtgataaact ggaaattagt caaggagatc attaagatcc accttaacac aagctttttt 3120 cgacttagga catgttcaaa gtctttaaga ctaataaaat ctaaacaact tggaaattct 3180 atcaaatttg gaaaaagatt tctaagctta tttaagcaag attttggaat tccacaagga 3240 tcaagtattt cttacattct ttgttgtctc tattataatt tcttagatct aaatccagaa 3300 atccagaatc ttttgggaca ttcatttttt tcctcctctt acatttcttt atcatttctg 3360 aatccaatta aacaagaaaa agtacaatta cttgatccaa ctgaaaaaga cttattttca 3420 caacatgatc ttgagacagg attatcccgt tacccagaat ttaaagaaaa tgagataaat 3480 atttataata ctagtacaag taaaaaaaga agactagaaa tatcagaata taatattaat 3540 gctaaaagtg ttttactgca accttccgaa gagtttaata ataatatcaa caaccagaat 3600 tgccaaatcc gtgccaatct caaaagttat gatcaagaaa gtccttctca gagcaataag 3660 caagaaagtc tcttattgag atgggtagac gattttcttt ttctcacatc cgatttggag 3720 tccgccaaaa agttcttaaa gttactttat atacaaaaac tatggggatc gaacgtttcc 3780 aaggacaaaa tcaactctaa ctttccttgg attgaccaca ataatgagat catcatcctt 3840 gaggaggatg atgttttact atcttcgcct tcctcttcct gttcatcatc ttgttcctct 3900 tcatcttcct cttcatcttc ttcgtcgtct tcctcatcgt cctcttcttc cttcccacct 3960 tcgacagagt gcacaagcag tccagtggaa aagagaataa ataccaacaa aagtgaaaag 4020 gactgccaaa atgaaaaaga aatgattaac aaggctaaga tagcctcaaa acagttccat 4080 aaacaagtat catggacagg aatgaagttt tccagtgagt acagctattt gaattgcatg 4140 gtttccccat ggaagaattt ggaattcata tgtgttatgg atactgtaac cttgacaact 4200 aagcaccaat ttacaagtaa ttattcaaat tttcacaggt ttaagtcgac gacgatatct 4260 gaaaaccttc aaaagtcaaa ttatatgtgg tctgttttgg gtataaaatt aataagatac 4320 tttgatttta gaatcaagaa tggattgcta tatgattgta aaatcaattc tttatttacg 4380 gtaagtttta aatataattc aacaaataac caaccaatta attaa 4425 59 960 PRT Giardia intestinalis 59 Met Asp Phe Pro Leu Ser Leu Leu Asp Ala Asn Val Ser Lys Ala Ile 1 5 10 15 Phe Ile Leu Leu Gln Gly Glu Pro Glu Gln Ala Ser Leu Trp Ser Leu 20 25 30 Ile Leu Gln Leu Arg Thr Pro Gly Gln Gly Ser Ser Phe Ala Ser Cys 35 40 45 Ala Ser Leu Ile Tyr Lys Asp Leu Lys Gly Leu Ser Ser Trp Ile Thr 50 55 60 Glu Ala Ala Phe Ile Pro Phe Leu Arg Arg Leu Gly Phe Cys Ser Leu 65 70 75 80 Tyr Leu Ser Tyr Gly His Asp Val Leu Leu Glu Leu Phe Lys Tyr Pro 85 90 95 Ile Val Leu Thr Ser Thr Val Ser Gly Lys Arg Val Leu Leu Ser Glu 100 105 110 Pro Gln Met Pro Leu Ala Phe Pro Pro Cys Leu His Asp Lys Ser Val 115 120 125 Ser Ser Met Val Arg Ala Ser Thr Val Gln Tyr Ala Ser Asn Cys Leu 130 135 140 Glu Glu Phe Ile Ser Met Ile Gln Glu His Pro Met Lys Leu Arg Phe 145 150 155 160 Ile Phe Phe Cys Lys Thr Arg Pro Tyr Ile Arg Ala Phe His Ser Leu 165 170 175 Ala Leu Leu Gly Ser Ala Thr Ala Ile Trp His Leu Ser Leu Leu Pro 180 185 190 Ser Gln Cys Leu Thr Ala Phe Tyr Gln Ala Asn Pro Leu Val Thr Phe 195 200 205 Lys Phe Pro Tyr Asn Ala Phe Ser Thr Arg Ser Phe Ser Ser Leu Leu 210 215 220 Gln Leu Cys Thr Ser Arg Gly Arg Leu Met Pro Asn Asn Ala Ile Asn 225 230 235 240 Ser Asn Ala Lys Ala Arg Ile Thr Arg Leu Ile Ser Ile Phe Arg Ser 245 250 255 Lys Met Lys Arg Leu Ser His Asn Leu Thr Phe Leu Lys Asp Lys Lys 260 265 270 Pro Gln Ser Leu Leu Gln Ser Phe Gly Phe Asp Thr His Phe Arg Val 275 280 285 Phe Leu Asp Glu Thr Gly Thr Leu Arg His Ala Pro Leu Pro Leu Ser 290 295 300 Val His Lys Ile Thr Leu Tyr Leu Met Leu Tyr Cys Lys Ser Ile Ser 305 310 315 320 Ala Phe Ser Ile Leu Gly Lys Arg Asn Ala Arg Thr Phe His Ser His 325 330 335 Leu Phe Arg Phe Ile Ser Gln Pro Met Asn Gly Phe Met Thr Leu Gln 340 345 350 Glu Leu Thr Ser Ser Ile Ser Thr Lys Glu Phe Leu Leu Pro Ala Thr 355 360 365 Thr Val Ser Lys Ala Glu Gly Glu Val Arg His His Leu Leu Ala Tyr 370 375 380 Phe Val Val Tyr Leu Met Asp Phe Val Ile Leu Pro Ala Leu Tyr Gln 385 390 395 400 Ser Phe Ser Ala Val Ser Met His Thr Glu Leu Ser Asn Arg Ile Glu 405 410 415 Thr Ala Asn Ser Met Val Pro Ala Leu Leu Ser Arg Ser Leu Trp Val 420 425 430 Ser Tyr Lys Asn Tyr Leu Ile Lys Ser Ser Ala Arg Leu Glu Ile Ala 435 440 445 Ala Thr Tyr Glu Phe Val Cys Ser Leu Ser Asn Leu His Ser Ile Pro 450 455 460 Leu Thr Asn Glu Asp Ala Val Asn Arg Ile Phe Trp Lys Gln Val Ser 465 470 475 480 Pro Gly Arg Leu Ser Phe Val Tyr Lys Ser Asn Gly Arg Val Arg Pro 485 490 495 Leu Cys Asn Phe Ser Phe Cys Ser Arg Arg His Arg Val Ser Leu Asn 500 505 510 Ser Phe Leu Arg Ser Ala Leu Gln Val Ile Ile Phe Glu Leu Glu Arg 515 520 525 Pro Arg Asn Arg Tyr Leu Gln Ala His Leu Ala Lys Asn Leu Gly His 530 535 540 Ile Ala Val Asp Tyr Ala Ala Trp Leu Asp Ala Thr Tyr Thr Glu Asn 545 550 555 560 Pro Glu Ala Gln Ile Tyr Met Val Thr Thr Asp Met Ile Thr Ala Phe 565 570 575 Glu Ser Val Ser Val Pro Arg Leu Leu Tyr Tyr Ile Ser His Tyr Ile 580 585 590 Val Thr Gly Asn Ala Tyr Val Val Leu Thr Tyr Lys Glu Ile Ala Pro 595 600 605 Ser Lys Met Pro Lys Ile Arg Thr Val Ala Leu Pro Cys Gln His Asn 610 615 620 Gly Cys Val Ser Met Asp Ser Ile Ser Arg Tyr Leu Leu Cys Lys Glu 625 630 635 640 Thr Pro Lys Lys Gln Pro Asn Ser Met Leu Cys Pro Met His Ala Arg 645 650 655 Ile Tyr Lys Arg Glu Asp Ile Ile Lys Met Leu Glu Leu His Leu Leu 660 665 670 Asn Pro Leu Val Ile His Glu Gly Gly Val Tyr Arg Leu Thr Ser Gly 675 680 685 Ile Pro Gln Gly Ser Val Val Ser Thr Val Leu Phe Asn Cys Tyr Ser 690 695 700 Ser Leu Leu His Val Gln Leu Leu Asn Thr Gly Ile Val Ser Ser Arg 705 710 715 720 Leu Phe Phe Tyr Arg Thr Phe Val Asp Asp Trp Leu Leu Leu Thr Thr 725 730 735 Ser Ile Ala Leu Leu Asp Ala Tyr Ile Glu Tyr Leu Asn Ile Met Arg 740 745 750 Asp His Gly Ala Tyr Phe Arg Leu Ser Cys Phe Thr Lys Pro His Lys 755 760 765 Asn Ser Pro Phe Ala Pro Ile Ala Thr Leu Asp Cys Ser Arg Glu Pro 770 775 780 Ile Pro Lys Lys Ser Leu Tyr Thr Pro Asn Leu Asn Gln Asp Met Tyr 785 790 795 800 Lys Leu Asp Val Pro Arg Tyr Cys Gly Tyr Ile Phe Leu Glu Asn Val 805 810 815 Ala Ile Ile Asp Val Leu Lys Trp Phe Ser Ser Thr Arg Thr Asn Gly 820 825 830 Ser Pro Ser Tyr Pro Met Ile Phe Gly Ser Gly Ile Ser Val Met Gln 835 840 845 Arg Pro Arg Leu Val Phe Lys Lys Arg Ile Arg Asp Leu Arg Cys Phe 850 855 860 Ile Glu Arg Ser Ile Gln Ala Pro Asp Pro Arg Ile Lys Val Asp Gly 865 870 875 880 Ser Ile Leu Tyr Ala Tyr Ile Arg Ala Val Thr Phe Cys Ile Leu Cys 885 890 895 Tyr Thr Arg Asn Ile Arg Tyr Ser Glu Ser Leu Arg Thr Leu Leu Arg 900 905 910 Ser Thr Phe Cys Ser Leu Lys Ala Thr Val Trp Arg His Ser Thr Ser 915 920 925 Arg Ala Ile Phe Ile His Val Ser Lys Arg Ile Ile Leu His Val Tyr 930 935 940 Gly His Asn Pro Arg Ile Arg Gly Leu Leu Leu Gly Met Leu Ser Arg 945 950 955 960 60 2883 DNA Giardia intestinalis 60 atggattttc cactgtctct tttggacgca aatgtcagca aggctatttt catcttgctt 60 caaggggagc ccgagcaagc ctccctatgg tcacttatcc ttcaactgcg aacccccggc 120 caggggtcca gcttcgcttc ttgcgcatcg ctgatttaca aagacctcaa gggacttagc 180 tcctggatta ccgaggctgc ttttattccc tttctcagac gtttgggatt ttgttcattg 240 tatctaagtt atgggcacga tgttctgctc gagcttttca aataccctat cgttctcact 300 tcgactgttt cagggaagcg ggtgctcctt tcagagcccc agatgcctct tgctttccca 360 ccttgcctcc atgacaaatc tgtatcatct atggttcgag cttccactgt gcagtatgct 420 tctaactgtc ttgaggaatt catttctatg attcaagagc accccatgaa gctacgattc 480 atattcttct gtaagacccg gccctacatc cgagcattcc acagcttggc attactagga 540 tcagcaacgg ccatctggca cctttctctt cttccttcac agtgccttac tgccttttat 600 caagccaatc cattagtcac atttaagttc ccgtacaatg ccttttccac gcgctcgttt 660 tcatctttgc tacagctatg cacatcccgt ggtcggctga tgccgaacaa tgctattaat 720 tcaaatgcta aagctagaat aacaagactc atttccattt ttcgtagtaa gatgaaacgg 780 ttaagccaca atcttacgtt tctgaaagac aagaaacccc aatctctttt gcaatcattt 840 ggctttgata cgcattttag ggtattctta gatgaaacag gcacccttcg tcacgcccct 900 ctgcccttat ctgtgcacaa aataactctt tatctcatgt tatattgcaa gtccatcagc 960 gcattttcta ttcttggcaa aaggaacgct cgaacatttc actcacatct ctttcgcttt 1020 atttctcagc caatgaacgg gtttatgacc ttgcaagagc tcacctctag tatctctaca 1080 aaagaattcc ttctcccagc aaccacagtc agcaaggccg agggagaggt gcgacatcac 1140 cttttagcgt acttcgttgt ctatctcatg gacttcgtca tacttcccgc cctctatcag 1200 tcgttttctg ctgtttctat gcacacggag ctttccaatc ggatcgagac ggccaactct 1260 atggttcctg cattgctttc ccgctcactt tgggtcagct acaagaatta cttgattaaa 1320 tcgtctgctc gtctagaaat agcggccact tatgaatttg tctgctcatt atccaatttg 1380 cactctatcc ctctaaccaa cgaagatgct gtgaaccgta tattttggaa gcaagtctct 1440 cccggtaggc tatcgtttgt ctataaatct aatggaaggg tgcggcccct atgtaacttt 1500 agtttttgta gccgacgaca cagggtctca cttaattcct ttcttcgaag cgcgctccag 1560 gtcatcatat ttgagctaga gcgcccacgc aataggtact tgcaagcaca tctagccaag 1620 aatctaggcc acatagcagt ggactatgct gcttggctcg atgcgacgta cacagaaaac 1680 cccgaggcgc agatatacat ggtcacaacc gatatgataa ctgcttttga aagcgtctcc 1740 gttcctcgct tactctacta catatcccat tacatcgtta ctgggaatgc ttacgttgtc 1800 ttgacttaca aggagattgc tcccagtaag atgcctaaga ttaggaccgt tgcgctgccc 1860 tgccagcata atgggtgtgt atccatggat tctatttccc gctaccttct ctgcaaggag 1920 acccctaaaa aacagcctaa ttctatgctg tgtcccatgc atgcacgaat ctataagcga 1980 gaggacatta tcaagatgct agagcttcat cttctcaatc ccctcgttat tcacgaagga 2040 ggagtatacc gtctgacatc tgggataccc cagggcagtg tcgtaagcac tgtccttttc 2100 aattgctatt cgtctctcct gcacgtgcaa ctactaaata cagggattgt ttcgtcccgt 2160 ctcttctttt acaggacctt tgttgacgac tggctgctat taactacgtc catagcatta 2220 cttgacgcat atatcgagta cctgaacatc atgcgtgatc atggagccta cttcagattg 2280 tcctgcttca cgaagccaca caaaaacagc ccattcgctc ccatagctac attagactgt 2340 tctagagaac ccataccaaa gaaaagtctt tacacaccca atctcaatca agatatgtac 2400 aaactagacg ttccacgata ttgtggatat atatttcttg aaaatgttgc cataatagac 2460 gtactgaagt ggttttcatc aacgcgaacc aatggcagtc cgtcctatcc catgattttt 2520 ggatctggga tatctgtcat gcagagaccc aggttggttt tcaaaaagag aatccgggat 2580 ctccgctgtt tcattgagag aagcatacag gcacccgatc cgcgtataaa agtggacggc 2640 tctattcttt atgcgtatat acgtgctgtt acgttttgca tactttgcta cacacgaaac 2700 ataagatact ctgagtcctt gcgcaccctt ttaaggagca cattctgttc tcttaaggcg 2760 accgtgtgga gacattctac atcccgtgct atatttatcc acgtatccaa acgcattatt 2820 ctacatgtct atggtcacaa tccccgcatc agagggcttc ttctgggaat gctaagcagg 2880 tag 2883 61 2629 PRT Mus musculus 61 Met Glu Lys Leu Cys Gly Tyr Val Pro Val His Pro Asp Ile Leu Ser 1 5 10 15 Leu Lys Asn Arg Cys Leu Thr Met Leu Ser Asp Ile Gln Pro Leu Glu 20 25 30 Lys Ile His Gly Gln Arg Ser Val Asn Pro Asp Ile Leu Ser Leu Glu 35 40 45 Asn Arg Cys Leu Thr Leu Leu Pro Asp Leu Gln Pro Met Glu Lys Ile 50 55 60 His Gly Gln Arg Ser Val His Pro Asp Ile Leu Ser Ser Glu Asn Arg 65 70 75 80 Cys Leu Thr Leu Leu Pro Asp Leu Gln Ser Leu Glu Lys Leu Cys Gly 85 90 95 His Met Ser Ser His Pro Asp Val Leu Ser Leu Glu Asn Arg Cys Leu 100 105 110 Ala Thr Leu Pro Thr Val Lys Arg Thr Val Ser Ser Gly Pro Leu Leu 115 120 125 Gln Cys Leu His Arg Ser His Thr Ala Gln Ala Asp Leu Arg Asp Pro 130 135 140 Asn Phe Arg Asn Cys Leu Phe Pro Glu Pro Pro Thr Ile Glu Ala Pro 145 150 155 160 Cys Phe Leu Lys Glu Leu Asp Leu Pro Thr Gly Pro Arg Ala Leu Lys 165 170 175 Ser Met Ser Ala Thr Ala Arg Val Gln Glu Val Ala Leu Gly Gln Arg 180 185 190 Cys Val Ser Glu Gly Lys Glu Leu Gln Glu Glu Lys Glu Ser Ala Glu 195 200 205 Val Pro Met Pro Leu Tyr Ser Leu Ser Leu Gly Gly Glu Glu Glu Glu 210 215 220 Val Val Gly Ala Pro Val Leu Lys Leu Thr Ser Gly Asp Ser Asp Ser 225 230 235 240 His Pro Glu Thr Thr Asp Gln Ile Leu Gln Glu Lys Lys Met Ala Leu 245 250 255 Leu Thr Leu Leu Cys Ser Ala Met Ala Ser Ser Val Asn Val Lys Asp 260 265 270 Ala Ser Asp Pro Thr Arg Ala Ser Ile His Glu Val Cys Ser Ala Leu 275 280 285 Ala Pro Leu Glu Pro Glu Phe Ile Leu Lys Ala Ser Leu Tyr Ala Arg 290 295 300 Gln Gln Leu Asn Leu Arg Asp Ile Ala Asn Ile Val Leu Ala Val Ala 305 310 315 320 Ala Leu Leu Pro Ala Cys Arg Pro His Val Arg Arg Tyr Tyr Ser Ala 325 330 335 Ile Val His Leu Pro Ser Asp Trp Ile Gln Val Ala Glu Phe Tyr Gln 340 345 350 Ser Leu Ala Glu Gly Asp Glu Lys Lys Leu Val Pro Leu Pro Ala Cys 355 360 365 Leu Arg Ala Ala Met Thr Asp Lys Phe Ala Gln Phe Asp Glu Tyr Gln 370 375 380 Leu Ala Lys Tyr Asn Pro Arg Lys His Arg Ser Lys Thr Arg Ser Arg 385 390 395 400 Gln Pro Pro Arg Pro Gln Arg Thr Lys Pro Pro Phe Ser Glu Ser Gly 405 410 415 Lys Cys Phe Pro Lys Ser Val Trp Pro Leu Lys Asn Glu Gln Ile Ser 420 425 430 Phe Glu Ala Ala Tyr Asn Ala Val Ser Glu Lys Lys Arg Leu Pro Arg 435 440 445 Phe Thr Leu Lys Lys Leu Val Glu Gln Leu His Ile His Glu Pro Ala 450 455 460 Gln His Val Gln Ala Leu Leu Gly Tyr Arg Tyr Pro Ser Thr Leu Glu 465 470 475 480 Leu Phe Ser Arg Ser His Leu Pro Gly Pro Trp Asp Ser Ser Arg Ala 485 490 495 Gly Gln Arg Met Lys Leu Gln Arg Pro Glu Thr Trp Glu Arg Glu Leu 500 505 510 Ser Leu Arg Gly Asn Arg Ala Ser Val Trp Glu Glu Leu Ile Asp Asn 515 520 525 Gly Lys Leu Pro Phe Met Ala Met Leu Arg Asn Leu Cys Asn Leu Leu 530 535 540 Arg Thr Gly Ile Ser Ala His His His Glu Leu Val Leu Gln Arg Leu 545 550 555 560 Gln His Glu Lys Ser Val Ile His Ser Arg Gln Phe Pro Phe Arg Phe 565 570 575 Leu Asn Ala His Asp Ser Leu Asp Arg Leu Glu Ala Gln Leu Arg Ser 580 585 590 Lys Ala Ser Pro Phe Pro Ser Asn Thr Thr Leu Met Lys Arg Ile Met 595 600 605 Ile Arg Asn Ser Lys Lys Ile Lys Arg Pro Ala Asn Pro Arg Tyr Leu 610 615 620 Cys Thr Leu Thr Gln Arg Gln Leu Arg Ala Ala Met Ala Ile Pro Val 625 630 635 640 Met Tyr Glu His Leu Lys Arg Glu Lys Leu Arg Leu His Lys Ala Arg 645 650 655 Gln Trp Thr Cys Asp Leu Glu Leu Leu Glu Arg Tyr Arg Gln Ala Leu 660 665 670 Glu Thr Ala Val Asn Ile Ser Val Lys His Asn Leu Pro Pro Leu Pro 675 680 685 Gly Arg Thr Leu Leu Val Tyr Leu Thr Asp Ala Asn Ala Asn Arg Leu 690 695 700 Cys Pro Lys Ser His Leu Gln Gly Pro Pro Leu Asn Tyr Val Leu Leu 705 710 715 720 Leu Ile Gly Met Met Met Ala Arg Ala Glu Gln Thr Thr Val Trp Leu 725 730 735 Cys Gly Thr Gly Thr Val Lys Thr Pro Val Leu Thr Ala Asp Glu Gly 740 745 750 Ile Leu Lys Thr Ala Ile Lys Leu Gln Ala Gln Val Gln Glu Leu Glu 755 760 765 Glu Asn Asp Glu Trp Pro Leu Glu Thr Phe Glu Lys Tyr Leu Leu Ser 770 775 780 Leu Ala Val Arg Arg Thr Pro Ile Asp Arg Val Ile Leu Phe Gly Gln 785 790 795 800 Arg Met Asp Thr Glu Leu Leu Asn Val Ala Lys Gln Ile Ile Trp Gln 805 810 815 His Val Asn Ser Lys Cys Leu Phe Val Ser Val Leu Leu Arg Lys Met 820 825 830 Gln Tyr Met Ser Pro Asn Leu Asn Pro Asn Asp Val Thr Leu Ser Gly 835 840 845 Cys Thr Asp Gly Ile Leu Lys Phe Ile Ala Glu His Gly Ala Ser Arg 850 855 860 Leu Leu Glu His Val Gly Gln Leu Asp Lys Ile Phe Lys Ile Pro Pro 865 870 875 880 Pro Pro Gly Lys Thr Lys Val Ser Pro Leu Arg Pro Leu Glu Glu Asn 885 890 895 Asn Pro Gly Pro Phe Val Pro Ile Ser Gln His Gly Trp Arg Asn Ile 900 905 910 Arg Leu Phe Ile Ser Ser Thr Phe Arg Asp Met His Gly Glu Arg Asp 915 920 925 Leu Leu Met Arg Ser Val Leu Pro Ala Leu Gln Ala Arg Ala Phe Pro 930 935 940 His Arg Ile Ser Leu His Ala Ile Asp Leu Arg Trp Gly Ile Thr Glu 945 950 955 960 Glu Glu Thr Arg Arg Asn Arg Gln Leu Glu Val Cys Leu Gly Glu Val 965 970 975 Glu Asn Ser Gln Leu Phe Val Gly Ile Leu Gly Ser Arg Tyr Gly Tyr 980 985 990 Thr Pro Pro Ser Tyr Asp Leu Pro Asp His Pro His Phe His Trp Thr 995 1000 1005 Gln Arg Tyr Pro Ser Gly Arg Ser Val Thr Glu Met Glu Val Met Gln 1010 1015 1020 Phe Leu Asn Arg Gly Gln Arg Ser Glu Pro Ser Asp Gln Ala Leu Ile 1025 1030 1035 1040 Tyr Phe Arg Asp Pro Gly Phe Leu Ser Ser Val Pro Asp Val Trp Lys 1045 1050 1055 Pro Asp Phe Ile Ser Glu Ser Glu Glu Ala Ala His Arg Val Ser Glu 1060 1065 1070 Leu Lys Arg Phe Leu Gln Glu Gln Lys Glu Val Thr Cys Arg Arg Tyr 1075 1080 1085 Ser Cys Glu Trp Gly Gly Val Ala Ala Gly Arg Pro Tyr Thr Gly Gly 1090 1095 1100 Leu Glu Glu Phe Gly Gln Leu Val Leu Gln Asp Val Trp Ser Val Ile 1105 1110 1115 1120 Gln Lys Arg Tyr Leu Gln Pro Gly Ala Gln Leu Glu Gln Pro Gly Ser 1125 1130 1135 Ile Ser Glu Glu Asp Leu Ile Gln Ala Ser Phe Gln Gln Leu Lys Ser 1140 1145 1150 Pro Pro Ser Pro Ala Arg Pro Arg Leu Leu Gln Asp Thr Val Gln Gln 1155 1160 1165 Leu Met Leu Pro His Gly Arg Leu Ser Leu Val Ile Gly Gln Ala Gly 1170 1175 1180 Gln Gly Lys Thr Ala Phe Leu Ala Ser Leu Val Ser Ala Leu Lys Val 1185 1190 1195 1200 Pro Asp Gln Pro Asn Val Ala Pro Phe Val Phe Phe His Phe Ser Ala 1205 1210 1215 Ala Arg Pro Asp Gln Cys Leu Ala Phe Asn Leu Leu Arg Arg Leu Cys 1220 1225 1230 Thr His Leu His Gln Lys Leu Gly Glu Pro Ser Ala Leu Pro Ser Thr 1235 1240 1245 Tyr Arg Gly Leu Val Trp Glu Leu Gln Gln Lys Leu Leu Leu Lys Ser 1250 1255 1260 Ala Gln Trp Leu Gln Pro Gly Gln Thr Leu Val Leu Ile Ile Asp Gly 1265 1270 1275 1280 Ala Asp Lys Leu Val Asp His Asn Gly Gln Leu Ile Ser Asp Trp Ile 1285 1290 1295 Pro Lys Ser Leu Pro Arg Arg Val His Leu Val Leu Ser Val Ser Ser 1300 1305 1310 Asp Ser Gly Leu Gly Glu Thr Leu Gln Gln Ser Gln Ser Ala Tyr Val 1315 1320 1325 Val Ala Leu Gly Ser Leu Val Pro Ser Ser Arg Ala Gln Leu Val Arg 1330 1335 1340 Glu Glu Leu Ala Leu Tyr Gly Lys Arg Leu Glu Glu Ser Pro Phe Asn 1345 1350 1355 1360 Asn Gln Met Arg Leu Leu Leu Ala Lys Gln Gly Ser Ser Leu Pro Leu 1365 1370 1375 Tyr Leu His Leu Val Thr Asp Tyr Leu Arg Leu Phe Thr Leu Tyr Glu 1380 1385 1390 Gln Val Ser Glu Arg Leu Arg Thr Leu Pro Ala Thr Leu Pro Leu Leu 1395 1400 1405 Leu Gln His Ile Leu Ser Thr Leu Glu Gln Glu His Gly His Asn Val 1410 1415 1420 Leu Pro Gln Ala Leu Thr Ala Leu Glu Val Thr His Ser Gly Leu Thr 1425 1430 1435 1440 Val Asp Gln Leu His Ala Val Leu Ser Thr Trp Leu Thr Leu Pro Lys 1445 1450 1455 Glu Thr Lys Ser Trp Glu Glu Ala Val Ala Ala Ser His Ser Gly Asn 1460 1465 1470 Leu Tyr Pro Leu Ala Pro Phe Ala Tyr Leu Val Gln Ser Leu Arg Ser 1475 1480 1485 Leu Leu Gly Glu Gly Pro Val Glu Arg Pro Gly Ala Arg Leu Cys Leu 1490 1495 1500 Ser Asp Gly Pro Leu Arg Thr Ala Val Lys Arg Arg Tyr Gly Lys Arg 1505 1510 1515 1520 Leu Gly Leu Glu Lys Thr Ala His Val Leu Ile Ala Ala His Leu Trp 1525 1530 1535 Lys Met Cys Asp Pro Asp Ala Ser Gly Thr Phe Arg Ser Cys Pro Pro 1540 1545 1550 Glu Ala Leu Lys Asp Leu Pro Tyr His Leu Leu Gln Ser Gly Asn His 1555 1560 1565 Gly Leu Leu Ala Lys Phe Leu Thr Asn Leu His Val Val Ala Ala Tyr 1570 1575 1580 Leu Glu Val Gly Leu Val Pro Asp Leu Leu Glu Ala Tyr Glu Leu Tyr 1585 1590 1595 1600 Ala Ser Ser Lys Pro Glu Val Asn Gln Lys Leu Pro Glu Ala Asp Val 1605 1610 1615 Ala Val Phe His Asn Phe Leu Lys Gln Gln Ala Ser Leu Leu Thr Gln 1620 1625 1630 Tyr Pro Leu Leu Leu Leu Gln Gln Ala Ala Ser Gln Pro Glu Glu Ser 1635 1640 1645 Pro Val Cys Cys Gln Ala Pro Leu Leu Thr Gln Arg Trp His Asn Gln 1650 1655 1660 Cys Ile Leu Lys Trp Ile Asn Lys Pro Gln Thr Leu Lys Gly Gln Gln 1665 1670 1675 1680 Ser Leu Ser Leu Pro Ile Ser Ser Ser Pro Thr Ala Val Ala Phe Ser 1685 1690 1695 Pro Asn Gly Gln Arg Ala Ala Val Gly Thr Ala Gly Gly Thr Ile Tyr 1700 1705 1710 Leu Leu Asn Leu Arg Thr Trp Gln Glu Glu Lys Ala Leu Val Ser Gly 1715 1720 1725 Cys Asp Gly Ile Ser Ser Phe Ala Phe Leu Ser Asp Thr Ala Leu Phe 1730 1735 1740 Leu Thr Thr Phe Asp Gly Leu Leu Glu Leu Trp Asp Leu Gln His Gly 1745 1750 1755 1760 Cys Trp Val Phe Gln Thr Lys Ala His Gln Tyr Gln Ile Thr Gly Cys 1765 1770 1775 Cys Leu Ser Pro Asp Arg Arg Leu Leu Ala Thr Val Cys Leu Gly Gly 1780 1785 1790 Tyr Val Lys Leu Trp Asp Thr Val Gln Gly Gln Leu Ala Phe Gln Tyr 1795 1800 1805 Thr His Pro Lys Ser Leu Asn Cys Ile Thr Phe His Pro Glu Gly Gln 1810 1815 1820 Val Val Ala Thr Gly Asn Trp Ser Gly Ile Val Thr Phe Phe Gln Ala 1825 1830 1835 1840 Asp Gly Leu Lys Val Thr Lys Glu Leu Gly Gly Pro Gly Pro Ser Val 1845 1850 1855 Arg Thr Leu Ala Phe Ser Ala Pro Gly Lys Val Val Ala Leu Gly Arg 1860 1865 1870 Ile Asp Gly Thr Val Glu Leu Trp Ala Trp Gln Glu Gly Thr Arg Leu 1875 1880 1885 Ala Ala Phe Pro Ala Gln Cys Gly Gly Val Ser Thr Val Leu Phe Leu 1890 1895 1900 His Ala Gly Gly Arg Phe Leu Thr Ala Gly Glu Asp Gly Lys Ala Gln 1905 1910 1915 1920 Leu Trp Ser Gly Phe Leu Gly Arg Pro Arg Gly Cys Leu Gly Ser Leu 1925 1930 1935 Tyr Leu Ser Pro Ala Leu Ser Val Ala Leu Asn Pro Asp Gly Asp Gln 1940 1945 1950 Val Ala Val Gly Tyr Arg Gly Asp Gly Ile Lys Ile Tyr Arg Ile Ser 1955 1960 1965 Ser Gly Pro Gln Glu Ala Gln Cys Gln Glu Leu Asn Val Ala Val Ser 1970 1975 1980 Ala Leu Val Trp Leu Ser Pro Ser Val Leu Val Ser Gly Ala Glu Asp 1985 1990 1995 2000 Gly Ser Leu His Gly Trp Met Leu Arg Arg Asn Ser Leu Gln Ser Leu 2005 2010 2015 Trp Leu Ser Ser Val Cys Gln Lys Pro Val Leu Gly Leu Ala Ala Ser 2020 2025 2030 Gln Glu Phe Leu Ala Ser Ala Ser Glu Asp Phe Thr Val Arg Leu Trp 2035 2040 2045 Pro Arg Gln Leu Leu Thr Gln Pro His Ala Val Glu Glu Leu Pro Cys 2050 2055 2060 Ala Ala Glu Leu Arg Gly His Glu Gly Pro Val Cys Cys Cys Ser Phe 2065 2070 2075 2080 Ser Pro Asp Gly Arg Ile Leu Ala Thr Ala Gly Arg Asp Arg Asn Leu 2085 2090 2095 Leu Cys Trp Asp Val Lys Val Ala Gln Ala Pro Leu Leu Ile His Thr 2100 2105 2110 Phe Ser Ser Cys His Arg Asp Trp Ile Thr Gly Cys Thr Trp Thr Lys 2115 2120 2125 Asp Asn Ile Leu Ile Ser Cys Ser Ser Asp Gly Ser Val Gly Leu Trp 2130 2135 2140 Asn Pro Glu Ala Gly Gln Gln Leu Gly Gln Phe Pro Gly His Gln Ser 2145 2150 2155 2160 Ala Val Ser Ala Val Val Ala Val Glu Glu His Ile Val Ser Val Ser 2165 2170 2175 Arg Asp Gly Thr Leu Lys Val Trp Asp Arg Gln Gly Val Glu Leu Thr 2180 2185 2190 Ser Ile Pro Ala His Ser Gly Pro Ile Ser Gln Cys Ala Ala Ala Leu 2195 2200 2205 Glu Pro Arg Pro Ala Gly Gln Pro Gly Ser Glu Leu Met Val Val Thr 2210 2215 2220 Val Gly Leu Asp Gly Ala Thr Lys Leu Trp His Pro Leu Leu Val Cys 2225 2230 2235 2240 Gln Ile His Thr Leu Gln Gly His Ser Gly Pro Val Thr Ala Ala Ala 2245 2250 2255 Ala Ser Glu Ala Ser Gly Leu Leu Leu Thr Ser Asp Asn Ser Ser Val 2260 2265 2270 Arg Leu Trp Gln Ile Pro Lys Glu Ala Asp Asp Thr Cys Lys Pro Arg 2275 2280 2285 Ser Ser Ala Val Ile Thr Ala Val Ala Trp Ala Pro Asp Gly Ser Leu 2290 2295 2300 Val Val Ser Gly Asn Glu Ala Gly Glu Leu Thr Leu Trp Gln Lys Ala 2305 2310 2315 2320 Gln Ala Val Ala Thr Ala Arg Ala Pro Gly Arg Val Ser Asp Leu Ile 2325 2330 2335 Trp Cys Ser Ala Asn Ala Phe Phe Val Leu Ser Ala Asn Glu Asn Val 2340 2345 2350 Ser Glu Trp Gln Val Glu Leu Arg Lys Gly Ser Thr Cys Thr Asn Phe 2355 2360 2365 Arg Leu Tyr Leu Lys Arg Val Leu Gln Glu Asp Leu Gly Val Leu Thr 2370 2375 2380 Gly Met Ala Leu Ala Pro Asp Gly Gln Ser Leu Ile Leu Met Lys Glu 2385 2390 2395 2400 Asp Val Glu Leu Leu Gln Met Lys Pro Gly Ser Thr Pro Ser Ser Ile 2405 2410 2415 Cys Arg Arg Tyr Ala Val His Ser Ser Ile Leu Cys Thr Ser Lys Asp 2420 2425 2430 Tyr Gly Leu Phe Tyr Leu Gln Gln Gly Asn Ser Gly Ser Leu Ser Ile 2435 2440 2445 Leu Glu Gln Glu Glu Ser Gly Lys Phe Glu Lys Thr Leu Asp Phe Asn 2450 2455 2460 Leu Asn Leu Asn Asn Pro Asn Gly Ser Pro Val Ser Ile Thr Gln Ala 2465 2470 2475 2480 Glu Pro Glu Ser Gly Ser Ser Leu Leu Cys Ala Thr Ser Asp Gly Met 2485 2490 2495 Leu Trp Asn Leu Ser Glu Cys Thr Pro Glu Gly Glu Trp Val Val Asp 2500 2505 2510 Asn Ile Trp Gln Lys Lys Ser Arg Asn Pro Lys Ser Arg Thr Pro Gly 2515 2520 2525 Thr Asp Ser Ser Pro Gly Leu Phe Cys Met Asp Ser Trp Val Glu Pro 2530 2535 2540 Thr His Leu Lys Ala Arg Gln Cys Lys Lys Ile His Leu Gly Ser Val 2545 2550 2555 2560 Thr Ala Leu His Val Leu Pro Gly Leu Leu Val Thr Ala Ser Glu Asp 2565 2570 2575 Arg Asp Val Lys Leu Trp Glu Arg Pro Ser Met Gln Leu Leu Gly Leu 2580 2585 2590 Phe Arg Cys Glu Gly Pro Val Ser Cys Leu Glu Pro Trp Met Glu Pro 2595 2600 2605 Ser Ser Pro Leu Gln Leu Ala Val Gly Asp Ala Gln Gly Asn Leu Tyr 2610 2615 2620 Phe Leu Ser Trp Glu 2625 62 1255 DNA Mus musculus 62 taacaacgtg caagtactat cacagtgcca aactagcaga gctgccatct aaggtcgagg 60 tcgccgcttt ggctgtgtgc acaggcaagc gccctcaccc aatggccctg gccttgctat 120 gggtgcgtga gttgagatga tgctctggac tctgaggtga aggccactgg aacagtgaaa 180 aaagctaacg cagggctttt acctagtccc cttcctttgg tggtgggtgt ttacggaaca 240 tatttgggat ctgagtgtat ggtcgcacca caataaagcc ttaacctata tagtagaatt 300 tcagctgtaa tcattaagaa ctgagattgc caccacccac ctcactgtct gtgtcaacca 360 cagcaggctg gagcagtcag ctcaggaaca ggcaaaacct taggtccctc cgcctaccta 420 accttcaata catcaaggat aggcttcttt gcttgcccaa acctcgcccc agtctagacc 480 acctggggat tcccagctca gggcgaaaag gaagcccgag aagcattctg tagagggaaa 540 tcctgcatga gtgcgccccc tttcgttact ccaacacatc cagcaaccac tgaacttggc 600 cggggaacac acctggtcct catgcaccag cattgtgacc atcaacggaa aagtactatt 660 gctgcgaccc cgccccttcc gatacaacgc ttggtccgcc tgaatcccgc cccttcctcc 720 gttcccagcc tcatcttttt cgtcgtggac tctcagtggc ctgggtcctg gctgttttct 780 aagcacaccc ttgcatcttg gttcccgcac gtgggaggcc catcccggcc ttgagcacaa 840 tgacccgcgc tcctcgttgc cccgcggtgc gctctctgct gcgcagccga taccgggagg 900 tgtggccgct ggcaaccttt gtgcggcgcc tggggcccga gggcaggcgg cttgtgcaac 960 ccggggaccc gaagatctac cgcactttgg ttgcccaatg cctagtgtgc atgcactggg 1020 gctcacagcc tccacctgcc gacctttcct tccaccaggt gggcctccag gcgggatccc 1080 catgggtcag gggcggaaag ccgggaggac atgggataat gcgtctagct catgtgtcaa 1140 gaccctcttc tccttaccag gtgtcatccc tgaaagagct ggtggccagg gttgtgcaga 1200 gactctgcga gcgcaacgag agaaacgtgc tggcttttgg ctttgagctg cttaa 1255 63 867 PRT Candida albicans 63 Met Thr Val Lys Val Asn Glu Lys Lys Thr Leu Leu Gln Tyr Val Leu 1 5 10 15 Asp Asn Thr Ser Asn Glu Val Pro Leu Leu Pro Ser Leu Lys Glu Tyr 20 25 30 Met Glu Thr Val Leu Val Tyr Gln Ser Ile Lys Arg Pro Leu Pro Ala 35 40 45 Ile Arg Pro Gln Glu Ser Phe Asp Glu Phe Met Lys Glu Leu Val Thr 50 55 60 Arg Leu Val Met Glu Lys Ser Asn Asn Val Ile Ala Tyr Gly Tyr Lys 65 70 75 80 Thr Ser Ala Met Glu Ser Arg Ser Ile Phe Thr Thr Phe His Ser Ser 85 90 95 Gly Asn Phe Ile Leu Thr His Ile Thr Ser His Asn Trp Ser Thr Ile 100 105 110 Phe Ser Leu Leu Gly Pro Lys Lys Phe Leu Glu Leu Leu Val Asn Asn 115 120 125 Lys Gly Phe Val Ser Lys Val Asn Gly Glu Ser Val Gln Ile Phe Gly 130 135 140 Asp Val Asn Ser His Arg Lys Ala Val Val Val Ser Lys Tyr Ile Thr 145 150 155 160 Lys Phe Asn Val Leu Tyr Asn Ser Tyr Ser Arg Asp Phe Ser Arg Phe 165 170 175 Glu Met Ile Arg Pro Ser Ile Gln Thr Ile Leu Gln Asp Ile Leu Ser 180 185 190 Phe Ser Gly Leu Asn Pro Gly Arg Ser Ser Lys Arg Tyr Arg Gly Phe 195 200 205 Lys Ser Leu Leu Ser Arg Ile Ile Ala Asn Asp Lys Lys Cys Arg Tyr 210 215 220 Asp Ile Leu Tyr Ala Lys Phe Ile Gly Thr Ser Lys Cys Asn Phe Ala 225 230 235 240 Asn Val Val Ser Asn Lys Thr Glu Ile Ser Gln Val Ile Gln Phe Val 245 250 255 Leu Leu Val Leu Gly Lys Leu Leu Pro Leu Asp Ala Trp Gly Gly Val 260 265 270 Ser Asn Lys Lys Ile Ile Lys Asp Arg Val Val Asp Phe Leu Leu Leu 275 280 285 Gly Ala Asn Glu Lys Ile His Met Asp Asp Leu Phe Arg Gly Ile Arg 290 295 300 Leu Lys Asp Phe Lys Trp Leu Gly Arg Ala His Gln Ile Ser Ser Lys 305 310 315 320 Gln Asp Phe Glu Leu Arg Thr Ala Phe Leu Lys Gly Tyr Leu Trp Trp 325 330 335 Leu Phe Glu His Leu Leu Lys Asn Ile Leu Arg Ser Phe Trp Tyr Ile 340 345 350 Thr Glu Thr Ser Ser Ile Val Ser Leu Glu Leu Asn Tyr Phe Pro Gln 355 360 365 Tyr Leu Trp Lys Glu Leu Tyr Glu Ser Trp Val Ser Lys Tyr Ala Lys 370 375 380 Asn Asn Leu Val Lys Met Pro Ser Lys Ile Gln Arg Glu Gln Leu Pro 385 390 395 400 Cys Gly Lys Ile Lys Leu Ile Pro Lys Arg Ser Ser Phe Arg Val Ile 405 410 415 Cys Val Pro Ile Lys Arg Ser Leu Lys Leu Leu Asn Lys Lys Leu Glu 420 425 430 Leu Asp Thr Leu Glu Lys Glu Lys Arg Glu Phe Glu Arg Tyr Arg Lys 435 440 445 Glu Val Leu Ser Pro Val Gly Gln Ile Leu Arg Leu Lys Leu Ser Lys 450 455 460 Leu Arg Asp Thr Tyr Glu Ser Tyr Arg Ala Ser Val His Ser Ser Ser 465 470 475 480 Asp Val Ala Glu Lys Ile Ser Asp Tyr Arg Asp Ser Leu Leu Thr Arg 485 490 495 Phe Gly Glu Ile Pro Lys Leu Phe Ile Leu Lys Phe Asp Met Lys Glu 500 505 510 Cys Tyr Asp Arg Leu Ser Gln Pro Val Leu Met Lys Lys Leu Glu Glu 515 520 525 Leu Phe Glu Asn Gln Asp Asn Lys Thr Ser Tyr Tyr Val Arg Tyr Tyr 530 535 540 Ala Gln Leu Asp Ala Ser His Lys Leu Lys Lys Val Lys Thr Thr Ile 545 550 555 560 Asp Thr Gln Tyr His Asn Leu Asn Ile Leu Ser Ser Ser Arg His Leu 565 570 575 Ser Asn Cys Lys Ser Leu Val Asp Lys Thr Lys Thr Ile Ala Leu Gln 580 585 590 Lys Gly Asn Ile Leu Glu Val Cys Arg Ser Gln Ile Tyr Asp Val Val 595 600 605 Gly Ser Val Lys Asp Ala Arg Gly Asn Leu His Leu Tyr Lys Arg Lys 610 615 620 Arg Gly Val Phe Gln Gly Phe Ser Leu Ser Ser Ile Phe Cys Asp Ile 625 630 635 640 Leu Tyr Ser Ala Met Val His Asp Cys Phe Gln Phe Leu Trp Lys Ser 645 650 655 Lys Gln Asp Phe Leu Phe Val Arg Leu Val Asp Asp Phe Leu Leu Val 660 665 670 Thr Pro Asp Ser Asn Ile Tyr Asp Gln Val His Asn Ile Leu Ser Gly 675 680 685 Lys Ile Leu Glu Ser Tyr Gly Ala Phe Val Asn Lys Asp Lys Thr Val 690 695 700 Val Val Asn Gln Thr Thr Thr Lys Pro Ser Ile Asp Phe Val Gly Leu 705 710 715 720 Glu Val Asn Thr Thr Asp Leu Ser Ile Lys Arg Asn Ser Gly Ser Ile 725 730 735 Ser Leu Val Thr Thr Asn Phe Arg Thr Phe Lys Thr Leu Val Lys Tyr 740 745 750 Leu Lys Thr Phe Tyr Gln Leu Asn Leu Glu Gly Phe Leu Leu Asp Cys 755 760 765 Ser Phe Gly Val Leu Glu Asn Val Leu Glu Asn Met Gly Ser Leu Leu 770 775 780 Arg Leu Val Leu Arg Glu Phe Lys Thr Lys Phe Thr Ser Ile Val Lys 785 790 795 800 Tyr Asp Thr Phe His Cys Tyr Lys Phe Ile Lys Phe Leu Tyr Asp Ile 805 810 815 Ser Asn Tyr Thr Ile Val Lys Tyr Val Glu Thr Asn Ser Asp Trp Asp 820 825 830 Gly Ala Pro Glu Leu Leu Asn Cys Ile Lys Gln Ile Ile Val Lys Glu 835 840 845 Phe Ser Ser Phe Glu Ser Tyr Ser Glu Ile Val Glu Trp Val Gln Thr 850 855 860 Leu Asn Ile 865 64 2714 DNA Candida albicans 64 cgttgttatt cacgcgtatc gtgagatatc atttcaaaga accacataca tgaccgtcaa 60 agtaaatgag aagaagactt tacttcagta tgttctagat aatacaagca atgaagtgcc 120 attgctacct agtttgaaag agtacatgga gacggtgctt gtataccaat ccataaaacg 180 gcctctacca gcgattcgac cacaagaatc atttgacgaa tttatgaaag agttggtgac 240 ccgtttagtt atggaaaaat cgaataatgt tatagcttat gggtataaga cctccgcaat 300 ggagagtcga agtatattta caacgtttca ttcgagtggg aattttattt taactcacat 360 tacaagccat aactggagta caatatttct gttactcgga cctaaaaaat ttctagagct 420 attagttaat aataaggggt ttgttagtaa ggtgaatggt gaatctgtgc aaatattcgg 480 tgacgtgaac tctcacagaa aggctgtcgt cgtttccaaa tacattacca aattcaatgt 540 gctttacaac tcctattcca gggacttctc acgctttgag atgataagac ccagtattca 600 aactatatta caggatattc tttccttttc tggtttgaat cctggaagat catccaaaag 660 atatcgaggc ttcaaaagtt tgctctcgag aattattgct aatgataaga aatgtagata 720 cgacattcta tatgctaagt ttattggtac gtcaaaatgc aattttgcta atgtggtgag 780 taataagaca gaaatatccc aggtaattca atttgtactt ttagtattgg gtaaattgtt 840 acctttggat gcttggggag gtgtttccaa taaaaagatt attaaggacc gagtggtaga 900 ttttttgtta cttggggcaa atgaaaagat acatatggat gatttattta gaggaattag 960 actaaaagat ttcaagtggt tgggcagagc tcaccaaatt tcttcgaaac aagatttcga 1020 gctccgaaca gcttttctaa aagggtatct atggtggttg tttgaacatt tacttaaaaa 1080 tattctccgt tctttctggt acattactga aacttcaagt atagtgagtt tagagttgaa 1140 ttattttcct cagtatttat ggaaagagct atacgagtca tgggtgtcta aatatgcaaa 1200 gaataatctt gtgaaaatgc catcaaagat ccaaagagaa caactaccat gtgggaaaat 1260 taaactcata cccaagcgct cgagctttcg tgttatttgt gtacctataa aacgatcctt 1320 gaaactattg aacaaaaaat tggaattgga cacattggaa aaggagaaaa gggaatttga 1380 aaggtacaga aaagaggttt tactgccagt gggacaaata ctacgcttga aattatcgaa 1440 actaagagat acatatgaaa gctatagggc ttcagtacat tccagttctg atgtggctga 1500 aaagatactg gattatagag actccttgtt aaccagattt ggcgaaatcc ctaagctttt 1560 catcttaaag tttgacatga aagaatgtta tgatagactc agccaacctg tattaatgaa 1620 aaaactagag gaacttttcg aaaaccaaga taataagact ctgtattatg ttcgatacta 1680 cgctcagttg gacgcgtcac ataaattgaa aaaagtgaaa accactatag atacccagta 1740 tcacaattta aacattttgt cgagctcaag gcatctcagt aattgtaaat ctttggtcga 1800 taagaccaag acaatagcgt tgcaaaaagg taacattttg gaagtttgtc gaagccaaat 1860 ctacgatgtt gttggttcag ttaaagatgc acgagggaat ttacacctat ataaaaggaa 1920 gaggggcgtg tttcagggat tctcattgct gtctatattt tgtgacatcc tatatagtgc 1980 aatggttcat gattgttttc aattcttatg gaagtcgaaa caggattttt tatttgtacg 2040 attggtagat gactttttac ttgtaacgcc cgattcgaat atttatgatc aagtgcacaa 2100 tatattatca ggaaaaatac ttgagagcta tggagctttt gttaataaag ataaaacagt 2160 cgttgttaat caaacaacca cgaaaccaag tatagatttc gttgggctcg aagtgaatac 2220 aacagatcta agcatcaaaa ggaactccgg tctgataagt ttggttacga caaacttcag 2280 aacattcaag actttagtta agtatttaaa gactttctat caattgaatt tggaggggtt 2340 tctcttggac tgttcttttg gggtattgga aaacgtgctt gaaaatatgg gatccctcct 2400 taggttggtt ttgagggaat tcaaaacaaa gtttacctcc attgtcaaat atgatacatt 2460 tcattgttac aaatttatca aatttctata tgacataagt aattacacaa tcgttaaata 2520 tgttgaaaca aacagcgact gggatggtgc acctgaacta ttgaattgca ttaaacagat 2580 aattgtcaag gagttttcct cttttgagag ttacctggaa atagtcgagt gggtacaaac 2640 attgaatata taaatacact gctcatatac ccccaaacga gctttttaaa ttctcgatat 2700 ctctcaattg tcgc 2714 65 34 PRT Artificial Sequence conserved amino acids in cdk4 p16Ink4a binding site 65 Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala Tyr Gly Thr Val Tyr 1 5 10 15 Lys Ala Arg Asp Xaa Xaa Ser Gly Xaa Phe Val Ala Leu Lys Xaa Val 20 25 30 Arg Val 66 11 PRT Artificial Sequence protein transport domain 66 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 67 11 PRT Artificial Sequence protein transport domain 67 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 68 34 PRT Artificial Sequence Consensus Sequence 68 Tyr Glu Xaa Val Ala Glu Ile Gly Xaa Gly Ala Tyr Gly Xaa Val Xaa 1 5 10 15 Lys Ala Arg Asp Xaa Xaa Xaa Gly Xaa Phe Val Ala Leu Lys Xaa Val 20 25 30 Arg Val 69 33 PRT Artificial Sequence Consensus Sequence 69 Xaa Xaa Xaa Val Xaa Xaa Ile Gly Xaa Gly Xaa Tyr Gly Xaa Val Tyr 1 5 10 15 Lys Ala Arg Xaa Xaa Xaa Xaa Gly Xaa Xaa Val Ala Leu Lys Xaa Xaa 20 25 30 Arg 

We claim:
 1. A composition for inducing a reversible state of continual growth in cultured cells, comprising at least one compound comprising a cdk4, cdk2 or cdk6 protein having an activating mutation, or biologically active fragment, derivative, homolog or analog of the cdk4, cdk2, cdk6 protein, wherein the compound further includes one or more modifications which allow the compound to enter the cells when administered to the cells in culture.
 2. The composition of claim 1, wherein the compound comprises a cdk4 protein selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 9; SEQ ID NO: 11; and SEQ ID NO: 13 having an activating mutation.
 3. The composition of claim 1 wherein the compound comprising the cdk4 protein comprises the amino acid sequence of SEQ ID NO:
 15. 4. The composition of claim 1, wherein the cdk4 protein activating mutation comprises an exchange of the conserved arginine in the p16^(Ink4a) binding site of the cdk4 protein for any amino acid.
 5. The composition of claim 4, wherein the conserved arginine is exchanged for a cysteine.
 6. The compound of claim 1, wherein the cdk4 protein comprises the amino acid sequence of SEQ ID NO: 27, and the activating mutation comprises a mutation in the amino acid sequence of SEQ ID NO:
 27. 7. The composition of claim 1, wherein the compound comprises a cdk6 protein selected from the group consisting of SEQ ID NO: 17; SEQ ID NO: 21; SEQ ID NO: 23; and SEQ ID NO: 25 having an activating mutation.
 8. The composition of claim 1, wherein the cdk6 protein activating mutation comprises an exchange of the conserved arginine in the p16 binding site in the cdk6 protein for any amino acid.
 9. The composition of claim 8, wherein the conserved arginine is exchanged for a cysteine.
 10. The compound of claim 1, wherein the cdk6 protein comprises the amino acid sequence of SEQ ID NO: 28, and the cdk6 activating mutation comprises a mutation in the amino acid sequence of SEQ ID NO:
 28. 11. The composition of claim 1, wherein the compound comprises a cdk4 protein or cdk6 protein comprising the amino acid sequence SEQ ID NO: 29, and the cdk4 or cdk6 activating mutation comprises a mutation in the amino acid sequence of SEQ ID NO:
 29. 12. The composition of claim 1, wherein the compound comprises a cdk2 protein selected from the group consisting of SEQ ID NO: 30; SEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 42; SEQ ID NO: 44; SEQ ID NO: 46; SEQ ID NO: 48; SEQ ID NO: 50; SEQ ID NO: 52; and SEQ ID NO: 54 having an activating mutation.
 13. The composition of claim 1 wherein the compound comprises a cdk2 protein which comprises the amino acid sequence of SEQ ID NO:
 32. 14. The composition of claim 1, wherein the compound comprises a cdk2 protein comprising the amino acid sequence SEQ ID NO: 55, and the cdk2 activating mutation comprises a mutation in the amino acid sequence of SEQ ID NO:
 55. 15. The composition of claim 1 further comprising a compound comprising a TERT protein, or biologically active fragment, derivative, homolog or analog thereof, wherein the compound includes one or more modifications which allow the compound to enter the cells when administered to the cells in culture.
 16. The composition of claim 15, wherein the compound is selected from the group consisting of SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 64; SEQ ID NO: 66; and SEQ ID NO:
 68. 17. The composition of claim 1, wherein said one or more modifications which allow the at least one compound to enter a cell comprise a leader sequence which directs entry of the compound into the cell.
 18. The composition of claim 17, wherein the leader sequence comprises SEQ ID NO: 70 or SEQ ID NO:
 71. 19. A method of inducing a reversible state of continual growth in cultured cells, comprising the steps of: a) providing a culture of viable cells; b) contacting the cells with an effective amount of the composition of claim 1; and c) optionally reversing the state of continual growth by removing the composition from contact with the cells.
 20. The method of claim 19, wherein the cells are obtained from amphibians, reptiles, birds, mammals, fish, arthropods or insects.
 21. The method of claim 20, wherein the cells are obtained from a mammal.
 22. The method of claim 21, wherein the mammal is selected from the group consisting of humans; rodents; rabbits; ovine mammals; bovine mammals; and porcine mammals.
 23. The method of claim 19, wherein the cultured cells comprise stem cells.
 24. The method of claim 23, wherein the stem cells are selected from the group consisting of hematopoietic stem cells; mesenchymal stem cells; neural stem cells; neural progenitor cells; embryonic stem cells; embryonic primordial germ cells; skeletal muscle satellite cells; and blastocyst inner cell masses.
 25. The method of claim 23 wherein the stem cells comprise mammalian stem cells.
 26. The method of claim 25 wherein the mammalian stem cells are mouse stem cells.
 27. The method of claim 25 wherein the mammalian stem cells are human stem cells.
 28. A method of screening an agent for the ability to transform cultured cells, comprising the steps of: a) providing a culture of viable cells; b) contacting the cells with an effective amount of the composition of claim 1, so that a state of continuous growth is induced for as long as the cells are in contact with the composition; c) contacting the cells with an agent; and d) evaluating the cells for the presence of a transformed phenotype.
 29. The method of claim 28, wherein the agent is selected from the group consisting of ionizing radiation, carcinogens, mutagens, teratogens, and nucleic acid sequences.
 30. The method of claim 28, wherein the nucleic acid sequences comprise a plasmid containing a gene or gene fragment.
 31. A cultured cell in which a reversible state of continual growth has been induced by the composition of claim
 1. 32. The cultured cell of claim 31, wherein the cells are obtained from amphibians, reptiles, birds, mammals, fish, arthropods, or insects.
 33. The cultured cell of claim 32, wherein the cells are obtained from a mammal.
 34. The cultured cell of claim 33, wherein the mammal is selected from the group consisting of humans; rodents; rabbits; ovine mammals; bovine mammals; and porcine mammals.
 35. The cultured cell of claim 31, wherein the cultured cells comprise stem cells.
 36. The cultured cell of claim 35, wherein the stem cells are selected from the group consisting of hematopoietic stem cells; mesenchymal stem cells; neural stem cells; neural progenitor cells; embryonic stem cells; embryonic primordial germ cells; skeletal muscle satellite cells; and blastocyst inner cell masses.
 37. The cultured cell of claim 35 wherein the stem cells comprise mammalian stem cells.
 38. The cultured cell of claim 37 wherein the mammalian stem cells are mouse stem cells.
 39. The cultured cell of claim 37 wherein the mammalian stem cells are human stem cells. 